A unique antigen on mature B cells defined by a monoclonal antibody

A novel 42,000 dalton antigen (MB-1) expressed by mature human B cells in blood and tonsil was identified and characterized by utilizing a hybridoma monoclonal antibody. A comparison of MB-1 with other known B cell antigens suggests that the MB-1 antigen has not been previously identified. From one-and two-color immunofluorescence studies, it appears that the MB-1 antigen is found on all normal immunoglobulin (Ig)-expressing cells, but not on T cells, thymocytes, granulocytes, or platelets. Studies of malignant B cell tumors reveal that the antigen is expressed by virtually all Ig-expressing B cell tumors but only 10% of SIg- B-lineage leukemias. Data from these studies suggest that the MB-1 antigen is expressed late in B cell ontogeny before the expression of SIg.     

Enhancement of human neutrophil functions by a monoclonal antibody directed against a 19-kDa antigen.

A mouse IgG mAb termed P1C3 was raised against A23187-treated human peripheral blood neutrophils and has been shown to recognize an Ag with an apparent molecular mass of 19 kDa, herein named p19. This p19 Ag was weakly expressed at the cell surface of resting human peripheral blood neutrophils and monocytes, but its cell surface expression was dramatically increased upon activation of these cell types with different secretagogues, including FMLP, PMA, and the calcium ionophores A23187 and ionomycin. A large latent pool of p19 molecules became accessible by immunofluorescence flow cytometry after cell permeabilization of resting neutrophils. A practically total translocation of the intracellular pool of this p19 molecule to the plasma membrane was achieved under appropriate cell stimulation, which induced an almost total degranulation of neutrophil secretory granules. The p19 Ag was absent from platelets, PBL, as well as from the human promyelocytic cell line HL-60, the human promonocytic cell line U937, and the human lymphoid cell lines Daudi and Jurkat. The p19 Ag was also expressed by circulating and/or interstitial neutrophils and monocytes in distinct tissues examined. The mAb P1C3 was found to enhance several neutrophil responses, such as chemotaxis, cell adhesion, phagocytosis, and respiratory burst. These data indicate that the mAb P1C3 recognizes an intracellular Ag in human resting mature neutrophils and monocytes, which upon cell activation is translocated to the cell surface and is able to affect cell functionality.     

Characterization of a novel rat thymocyte costimulating antigen by the monoclonal antibody 1.3.

To define membrane-associated molecules that impart signals for the activation and expansion of double negative (DN) cells, mAb were raised against in vitro-cultured rat DN cells. One such mAb, 1.3, stimulated proliferation of DN cells along with submitogenic concentrations of PMA and IL-2 without affecting the mobilization of Ca2+. The 1.3 mAb precipitated a heterodimeric protein from DN cells and kidney (130/110 kDa). Although the tissue distribution and biochemical characteristics of the 1.3 determinant resemble the neutral aminopeptidase (AP-N) first described as the thymocyte activating molecule in the mouse, other data are contradictory; AP-N message was not detected in mRNA from 1.3 positive cells and the AP-N gene was absent in the genomic DNA from rat DN hybridomas expressing high levels of 1.3 Ag. In addition, the 1.3 mAb did not affect AP-N enzyme activity suggesting that 1.3 mAb does not function through this enzyme to transduce signals for proliferation. Thus, the 1.3 mAb defines a new and important thymocyte costimulating Ag.     

Novel monoclonal antibody mAb G3A5 recognizes 138-kDa glycoprotein localized on the Golgi membrane.

Partially purified Golgi membranes of HeLa cells were used as antigen to produce a novel monoclonal antibody (mAb G3A5). The mAb G3A5 specifically labeled Golgi apparatus of human and monkey cultured cells as ascertained by indirect immunofluorescence but did not stain those of bovine or mouse cells. Treatment with nocodazole and brefeldin A (BFA) induced fragmentation and redistribution of the staining. Western immunoblot analysis showed that mAb G3A5 was directed against a single polypeptide with an apparent molecular mass of 138-kDa (p138 antigen). The p138 antigen is an integral membrane protein of the Golgi apparatus, as assessed by several assays: protease protection, salt wash and flotation in sucrose density gradient centrifugation. The p138 antigen was purified using immunoaffinity chromatography. The apparent molecular mass of the p138 antigen decreased by 2 to 4 kDa after treatment with the peptide: N-glycosidase F, while digestion with ENDO F or Neuraminidase did not have this effect. Thus, p138 antigen is a glycoprotein containing asparagine-linked carbohydrates.     

Human monoclonal antibodies to a genus-specific chlamydial antigen, produced by EBV-transformed B cells

Stable B cell lines producing human monoclonal antibodies to Chlamydia were established from salpingitis patients in the early convalescence phase. The antibody-producing cells were immortalized by Epstein Barr virus (EBV) transformation. Specific antibody-secreting clones were enriched by a stepwise microtiter plate cloning procedure. The selected B cell clones showed stable antibody production for more than 1 yr in continuous culture. Serologic specificity was demonstrated by micro-immunofluorescence (micro-IF) tests against a panel of Chlamydia reference strains. The antibodies were of the IgG1 subclass, and complement fixation could be demonstrated for one clone. There was no cross-reactivity against a large number of other bacteria. The monoclonal antibodies are directed against a common genus-specific surface antigen of the Chlamydia organism. Infected McCoy cells showed a brilliant, punctuated fluorescence surrounded by an inclusion membrane. Compared with conventional antisera, the monoclonal antibodies showed a clearer fluorescence pattern with very low background.     

Trichinella spiralis: a 76-kDa excretory/secretory larval antigen identified by a monoclonal antibody

Spleen cells from BALB/c mice were fused with myeloma cells following infection of the mice with Trichinella spiralis larvae and an ip booster injection with larval homogenate antigen. A monoclonal antibody (Mab), designated as TS 3G6 which did not react with sera or tissue extracts from noninfected mice, rats, and guinea pigs, was selected for further studies because of its high activity and specificity. When tested in ELISA TS 3G6 did not cross-react with Ascaris suum, A. lumbricoides, Toxocara canis, E. granulosus (larvae), Trichiuris suis, or T. ovis. Western blot analysis showed that Mab 3G6 recognized an antigen of 76 kDa located in the stichosome of the larvae as well as on the surface of the larval cuticle. Digestion of a larval extract with different enzymes suggests that the Mab TS 3G6 corresponding epitope is a polypeptide. The TS 3G6 antigen was detected in culture supernatants of Trichinella muscle larvae and in sera of experimentally infected animals using a sensitive ELISA assay. This secretory antigen also seemed to induce a specific immune response in the host since sera from infected animals could block the binding of Mab TS 3G6 to its target antigen when tested in a competitive ELISA.     

Selection of an attenuated Coxsackievirus B3 variant, using a monoclonal antibody reactive to myocyte antigen.

Previously, we described a heart-reactive monoclonal antibody (MAb), 10A1, derived from a coxsackievirus B3 (CVB3)-infected mouse. This MAb selectively inhibits infection of HeLa cells and myocytes with the myocarditic virus variant (CVB3W). A plaque-purified variant (H3) of CVB3W was isolated from the heart of an infected animal, and a second virus (H3-10A1) was obtained by growing H3 in HeLa cells in the presence of MAb 10A1. As with the parental CVB3W virus, H3 infection of HeLa cells can be inhibited by MAb 10A1, but the antibody-selected H3-10A1 variant is resistant to MAb inhibition (presumably an escape mutant). BALB/c mice infected with 10(6) PFU of CVB3W, H3, or H3-10A1 resulted in approximately 90% animal mortality with CVB3W or H3 and less than 10% mortality with H3-10A1, suggesting that the escape mutant is less pathogenic. Additionally, hearts from animals infected with H3-10A1 demonstrated only half the amount of myocarditis observed in either CVB3W- or H3-infected mice. Cardiac virus titers were also reduced approximately 200-fold in H3-10A1-infected animals compared with those in mice given the pathogenic variants. In vitro studies indicate that H3-10A1 is less effective in inhibiting cellular RNA and protein synthesis and show reduced virus replication compared with that of pathogenic viruses in cultured myocytes.     

Human milk bile-salt-stimulated lipase is extremely reactive with the monoclonal antibody 1CF11 which recognizes a human-specific carbohydrate antigen

1CF11 (Kanamaru, Y. et al.; Biochem. Biophys. Res. Commun., 249, 618-623, 1998) is a monoclonal antibody obtained after being raised in a mouse by injection of human milk MUC1 mucin as the antigen. Its reactivity was found to be unique in that it only reacts with a carbohydrate epitope shared by glycoproteins in human secretions, while its chemical nature is still unknown. Since a glycoprotein of Mr 135,000 (135K) in human milk was found to react extremely strongly with this antibody, we intended in this study to isolate the glycoprotein by a combination of various chromatographic techniques and identify it. It is a human milk bile-salt-stimulated lipase. By comparison of its immunoreactivity and glycan structures so far reported with those of lactoferrin from human milk, it is suggested that the epitope recognized by mAb ICF11 could be a human-specific novel glycan.     

A monoclonal antibody reactive with a 40-kDa molecule on fetal thymocytes and tumor cells blocks proliferation and stimulates aggregation and apoptosis.

E710.2.3 is a murine thymic lymphoma cell line with an immature phenotype (CD4-CD8-) that proliferates in response to thymocytes or PMA when cultured at low density and proliferates spontaneously when grown at high density. To identify functional molecules on this cell line, we screened for mAbs that could block its proliferation. A hamster mAb, DMF10.62.3, inhibited the spontaneous, thymocyte-induced, and PMA-stimulated proliferation of E710.2.3 in vitro and induced these cells to undergo apoptosis. The mAb also caused homotypic aggregation of E710.2.3, which was inhibited by cytochalasin B, trifluoperazine, a combination of sodium azide and 2-deoxyglucose, EDTA, incubation at 4 degrees C, or treatment with paraformaldehyde. The DMF10 62.3 mAb stained a number of immortalized murine and human cell lines and, where tested, blocked their proliferation and caused death to varying extents by apoptosis. The molecule recognized by the mAb DMF10.62.3 was expressed on day 14 fetal thymus Thy1.2-positive cells. However, it was not detected on adult murine thymocytes, splenocytes, or bone marrow cells or on splenic LPS-activated B cells or Con A-activated T cells. The Ab immunoprecipitated a 40-kDa molecule from E710.2.3 that was not glycosylphosphatidylinositol linked. The data suggest that the molecule recognized by DMF62.3 is a novel cell surface molecule that may be involved in cell proliferation and/or cell death.     

Cell type specificity of a neural cell surface antigen recognized by the monoclonal antibody A2B5

Summary The monoclonal antibody A2B5 reacts with the surface membrane of most neurons in monolayer cultures of cerebellum, retina, spinal cord, and dorsal root ganglion of embryonic and early postnatal C57BL/6J mice maintained in vitro for culture periods of 2 to 10 days. A small percentage of astroglial cells also expresses A2B5 antigen in murine, chicken and rabbit cerebellum, in chicken retina, and in murine spinal cord and dorsal root ganglion. Less mature astroglial cells are stained for A2B5 antigen to a greater extent than the more mature astrocytes. Astrocytes from rat cerebellum and mouse retina were not found to express A2B5 antigen under the present culture conditions. Some of the less mature oligodendrocytes recognized by 04 antibodies express A2B5 antigen, while the more mature 01 antigen and galactocerebroside-positive oligodendrocytes were not found to be A2B5 antigen-positive. Fibroblasts or fibroblast-like cells do not express detectable levels of A2B5 antigen. After fixation of the cells with paraformaldehyde and ethanol, all cell types present in culture are labeled by the A2B5 antibody intracellularly.     

Inhibition of adhesion of F9 embryonal carcinoma cells to substratum by a novel monoclonal antibody, TEC-05, reactive with a developmentally regulated carbohydrate epitope

Embryonal carcinoma cells carry on their surfaces carbohydrate antigens that are also expressed in early embryonic cells. We report here the expression and properties of a new developmentally regulated carbohydrate epitope, which is defined by a monoclonal antibody TEC-05. This antibody was generated by immunization of a rat with mouse embryonal carcinoma cells P19S1801A1. By immunofluorescence, the TEC-5 epitope was first detected on 8-cell-stage mouse embryos and was present on all subsequent stages of preimplantation development. Absorption analysis revealed that TEC-5 epitope was expressed only on a limited number of adult mouse tissues. In the direct radioantibody binding assay, TEC-05 reacted strongly with OTF9-63 cells and with some of the mouse embryonal carcinoma cell lines tested. Its reaction with differentiated cell lines was weak or undetectable. In the course of differentiation of OTF9-63 cells induced by retinoic acid, the epitope disappeared with the onset of morphological differentiation. The binding of the antibody to OTF9-63 cells was inhibited to 50% by 10-50 microM N-acetyllactosamine and lactose. Immunolabelling of extracts from OTF9-63 cells separated by sodium-dodecyl-sulfate (SDS) polyacrylamide gel electrophoresis revealed that TEC-5 epitope was carried by high-molecular-weight glycoconjugates (molecular weight greater than 100,000). Molecules, isolated from [3H]-fucose-labelled OTF9-63 cells by indirect immunoprecipitation with TEC-05 antibody, were degraded by extensive pronase digestion or mild alkaline treatment to large carbohydrate chains that were excluded from a Sephadex G-50 column. Direct evidence that TEC-05 antibody bound to embryoglycan was obtained using a modified Farr's assay. The antibody was found to inhibit adhesion of F9 and OTF9-63 cells to substratum. The inhibitory effect, which could be abrogated by lactose, seemed to be specific, because another IgM monoclonal antibody which also binds to embryoglycan had no effect. Combined data indicated that TEC-05 antibody recognizes a carbohydrate epitope which is involved in cell-substratum adhesion of F9 cells and which provides a new marker for structure-function studies of stage-specific embryonic antigens.     

Sialosyllactotetraosylceramide, a novel ganglioside antigen detected in human carcinomas by a monoclonal antibody

A novel ganglioside was detected in a small cell lung carcinoma by TLC-immunostaining of gangliosides with a monoclonal antibody, the C-50 MAb. Structural characterization showed this ganglioside to be IV3NeuAc-LcOse4Cer, a hitherto unknown ganglioside. This ganglioside has also been detected as a minor component in many different carcinomas using the C-50 MAb. The normally dominant CA-50 ganglioside antigen is IV3NeuAc, III4Fuc-LcOse4Cer. Based upon solid-phase binding to IV3NeuAc, III4-LcOse4Cer and IV3NeuAc-LcOse4Cer it is concluded that the C-50 MAb recognizes an epitope present in sialylated type I carbohydrate chains.     

5C6-F4, a novel 100,000-dalton rat lymphocyte activation antigen defined by monoclonal antibody

Con A-activated rat thymocytes were used to immunize mice, and immune spleen cells were fused with NS/1 myeloma cells. One clone, designated 5C6-F4, reacted strongly with Con A-activated rat thymocytes and some LPS-activated rat spleen cells but not with normal thymocytes, spleen cells, or bone marrow cells of rat origin. The 5C6-F4 did not react with Con A-activated thymocytes of mouse origin. Immunoprecipitation of 5C6-F4 antigen from surface-iodinated Con A-activated rat thymocytes or LPS-activated rat spleen cells revealed its m.w. to be approximately 100,000. The kinetic studies of the expression of 5C6-F4 antigen revealed that 5C6-F4 antigen was detectable at 6 hr after Con A stimulation of rat spleen cells, whereas IL 2 receptor (IL 2R) was detectable at 12 hr. The appearance of 5C6-F4 antigen and IL 2R precede the onset of DNA synthesis of Con A-activated spleen cells. Thus, 5C6-F4 antigen is classified as early activation antigen. The 5C6-F4 inhibits the lymphocyte proliferation induced by mitogen and the IL 2-driven rat T cell proliferation. Sequential immunoprecipitation study as well as binding inhibition study indicated that the 5C6-F4 antigen is distinct from IL 2R molecule. The 5C6-F4 antigen appears to be a novel rat lymphocyte activation antigen that exhibits immunoregulatory function and also may serve as a useful marker of T cell activation.     

Cloning and sequencing of a 24-kDa Trypanosoma cruzi specific antigen released in association with membrane vesicles and defined by a monoclonal antibody.

In the present study we have used the Tcr7 monoclonal antibody (mAb) previously characterized as directed against Trypanosoma cruzi 24-25-kDa specific antigens, both are immunogenic in man and during experimental T cruzi infections. We have demonstrated that mAb Tcr7 was able to recognize two in vitro translation products of molecular weights of 24 and 25 kDa. This suggested the holoproteic nature of these two related antigens bearing at least a common epitope and allowed us to use Tcr7 for an immunoscreening of a lambda ZAPII T cruzi cDNA library. Indeed, we have obtained several positive clones and completely sequenced the largest one which encoded theoretically for a protein of 23.7 kDa. The sequence analysis revealed a nearly perfect homology between this clone and one already described by other investigators and was shown to express a major flagellar protein of T cruzi able to bind calcium. Using different overlapping peptides derived from the sequence of the cDNA clone, we have localized the immunoreactivity of mAb Tcr7 mainly on several primary sequences present in the N-terminal part of the sequence, suggesting that the mAb could recognize a discontinuous epitope. Moreover, the immunoelectron microscopy allowed us to show that the antigen(s) carrying the epitope reacting with mAb Tcr7 is (are) released in association with membrane vesicles which protruded from the parasite surface and the flagellar pocket. This new mechanism of antigen shedding is likely to be independent of phospholipase C-mediated release of GPI-anchored molecules.     

Identification of a 220-kDa membrane tumor-associated antigen by human anti-UK114 monoclonal antibodies selected from the immunoglobulin repertoire of a cancer patient

Human monoclonal antibodies (HuMAb) specific for a 14-kDa perchloric acid-soluble protein (defined as UK114) were produced by somatic fusion of the human-mouse myeloma K6H6/B5 with Epstein-Barr virus-transformed peripheral B lymphocytes from a cancer patient previously treated with UK101 preparations, containing the UK114 protein. Three IgM-secreting clones were selected on the criteria of specificity for the purified UK114 protein immobilized onto plastic and adapted to grow in a serum-free medium. The reactivity of these antibodies showed a broad distribution pattern restricted to fresh tumor tissues and tumor cell lines, mainly of the adenocarcinoma type. None of the normal cells, nonmalignant cell lines, and normal tissues surrounding the neoplastic lesions were reactive. The immunochemical analysis of the target antigens showed that the HuMAb recognize a molecule of 220 kDa selectively expressed by the surface of tumor cells, as well as a cytoplasmic 14-kDa protein. The 220-kDa antigen was different from other tumor-associated antigens with similar molecular mass and, so far, unique. In the presence of human complement, two of three HuMAb are cytotoxic for tumor cells expressing the 220-kDa surface antigen. The tumor specificity and the lytic ability attributed to these HuMAb are promising features for the exploration of future clinical applications.     

A monoclonal antibody against a novel 20-kDa protein induces cell adhesion and cytoskeleton-dependent morphologic changes.

A new murine IgA mAb (JKT.M1), developed against Jurkat T cells chronically infected with HIV IIIB induces in vitro homotypic aggregation in several hemopoietic cell lines. The JKT.M1 Ag is expressed on a wide variety of cell types including human lymphocytes, monocytes, platelets, RBC, human umbilical vein endothelial cells, many T cell lines, myelomonocytic cell lines, and a primate kidney cell line. The JKT.M1 Ag shows differential expression on myelomonocytic cells; it is present on K562 and HL60 cell lines, which represent precursors of E and monocytes, respectively, but is not expressed on the surface of U937 and THP-1 cell lines, which appear to represent intermediate cell types of the monocytic cell lineage. However, the JKT.M1 Ag is expressed on mature peripheral blood monocytes and the MonoMac cell line. Immunoprecipitation from cell lysates (Jurkat, SupT1, PBMC, MonoMac) with the JKT.M1 mAb yields a 20-kDa Ag with few if any carbohydrate residues as determined by N-glycanase and neuraminidase treatments. The pI appears acidic by two-dimensional gel analysis, and the nonreduced form migrates more slowly than the reduced form when analyzed by SDS-PAGE suggesting the presence of intramolecular disulfide bridge(s). JKT.M1 mAb-induced cell adhesion is shown to be divalent cation- and temperature-dependent. The adhesion induced by JKT.M1 mAb is inhibited by 20 microM cytochalasin B and also by 2 mM 2-deoxyglucose plus 10 mM sodium azide suggesting that cytoskeletal changes and metabolic energy are required. Aggregation induced by JKT.M1 appears to be independent of CD43, CD44, and VLA4 (CD29/CD49d), mAb against which have also been shown to induce homotypic cell adhesion. Anti-CD18 mAb have been shown to inhibit homotypic aggregation in other studies but failed to do so in the present study. Thus JKT.M1-induced adhesion also appears to be independent of CD18, the beta-chain of leukocyte integrins. However, like mAb against LFA-1, immobilized JKT.M1 stimulates a T cell line to undergo dramatic morphologic changes which could be enhanced by the addition of phorbol ester. These data suggest that the novel 20-kDa molecule recognized by the JKT.M1 mAb may trigger cell adhesion through a previously undescribed mechanism.     

Human T and B cell immunoreactivity to a recombinant 23-kDa Cryptosporidium parvum antigen.

Cryptosporidial infection in humans results in parasite-specific IgG, IgM, and IgA antibody responses, but little is known of the cell-mediated immune responses to cryptosporidial antigens. In a convenience sample of 35 Haitian residents, there was a high level of cryptosporidial exposure (>90%) as determined by immunoblot reactivity of serum against cryptosporidial antigens. An attempt was made to determine if there was a relationship between antibody and T cell-mediated responses to recombinant Cp23 antigen and how this correlated with reactivity to crude sporozoite antigen preparations (SAg). T cell reactivity was greater against SAg (57%) than to Cp23 (34.3%) as measured by [3H]thymidine incorporation. Proliferative responses to Cp23 were significantly correlated with SAg responses. By enzyme-linked immunosorbent assay, most persons had IgG responses to both SAg (91.4%) and to recombinant Cp23 (88.5%). Antibody responses were greater among persons who exhibited T cell responses to SAg and Cp23. This study demonstrates that recombinant Cp23 antigen could be a useful antigen for detection of both antibody and cell-mediated responses in epidemiologic studies.     

A monoclonal antibody to the p220 component of sheep LCA identifies B cells and a unique lymphocyte subset

The leukocyte common antigen (LCA, CD45) of humans and rodents is expressed exclusively by leukocytes, and has been implicated in a number of immune functions (1-4), although its precise function is still unknown. Three monoclonal antibodies (mAbs) were produced which identified different epitopes on the LCA of sheep. mAbs 1-11-32 and 38-42 reacted with determinants of LCA expressed on all leukocytes, but showed differential reactivity with thymocytes. Another antibody, 20-96, identified an epitope of LCA expressed mainly on B cells, but also on a unique lymphocyte subset contained mostly in peripheral blood, which was 20-96high, sIg-, CD4-, CD8-, SBU-T19-, but CD5+. These cells constituted only 5-6% of PBL. The cellular lineage of this latter subset is uncertain since these cells appeared to be unrelated to B cells, and were absent from the thymus. Unlike the other two mAbs to LCA, 20-96 was not reactive with macrophages and granulocytes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of material immunoprecipitated by the "pan" LCA-specific mAbs revealed lymphocyte forms with molecular weights (MW) of 220, 210, and 190K, whereas 20-96 immunoprecipitated only a 220K MW form. The expression and MW of LCA on thymocytes or ileal Peyer's patch (IPP) cells differed from those on peripheral lymphocytes. B cells in IPP, which constitute 98% of cells, expressed the 20-96 determinant at low density, in contrast to its high expression on peripheral B cells. LCA from IPP existed in two forms of 220 and 190K MW, whereas LCA from peripheral B cells was entirely 220K MW, and thymus 210 and 190K MW.     

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.     

A novel fibrillar structure in cultured cells detected by a monoclonal antibody

Using a monoclonal antibody, we have detected an antigen present in a unique fibrillar structure in the cytoplasm of cultured cells by immunofluorescence. These structures have been identified by transmission electron microscopy and ultrastructural immunocytochemistry as large single paracrystalline arrays of individual filaments morphologically similar to intermediate filaments. The antibody detects these structures in fibroblastic and epithelioid cultured cell lines of mouse, rat, bovine, and human origin but not of avian origin. Only a small percentage of the cells in a culture contains these structures; each cell usually contains only one, although two or more have been observed in a single cell. The structures are elongated vermiform arrays of filaments in the cytoplasm (approximately 0.5 X 3 microns) which have a thread-like or toroidal appearance. Because of this shape, we have named the putative antigen recognized by this antibody "nematin." Double-label experiments showed that these structures had no relationship to tubulin or vimentin. Immunocytochemical localization in human tissues revealed a high concentration of a reactive antigen in the stratum granulosum of skin and in what probably are neuroglial cells in the central nervous system. This monoclonal antibody may detect a novel intermediate filament protein and/or a shared determinant of different intermediate filament proteins.