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R T Kubo

Publications and source records attributed to R T Kubo.

At least 73 records · Page 4Linked to original sources
PubMed1988

Activation induces a rapid reorganization of spectrin in lymphocytes.

Lymphocyte activation results in a rapid reorganization of the cytoskeletal protein spectrin. Immediately following an activation signal, there is fragmentation of a spectrin-rich cytoplasmic structure and subsequent translocation of the fragments to defined areas of the plasma membrane in both antigen-specific T cell hybridomas and lymph node T cells. These dramatic changes have been documented by light and electron microscopic immunolocalization and by immunoblot analysis of plasma membrane-enriched preparations. A T cell hybridoma variant lacking the spectrin-rich cytoplasmic structure of the parental line does not redistribute spectrin and produces little or no IL-2 in response to antigen-dependent activation. This suggests a functional link between spectrin distribution and activation potential. We propose that the cytoplasmic structure functions as an organizing center or reservoir for spectrin that is sensitive to signaling at the cell surface.

Animals↗
PubMed1988

Soluble factors in tolerance and contact sensitivity to 2,4-dinitro-fluorobenzene in mice. IX. A monoclonal T cell suppressor molecule is structurally and serologically related to the alpha/beta T cell receptor.

Ts cells from mice tolerized with dinitrobenzene sulfonate produce a DNP-specific, MHC-restricted soluble suppressor factor (SSF) which regulates contact sensitivity to 2,4-dinitro-fluorobenzene. Previous studies have shown that the SSF-producing T cells and the soluble factor have the same hapten/MHC specificity suggesting that SSF may represent a secreted form of the Ts membrane receptor. The relationship between TCR proteins and SSF was investigated by examining the structural and serologic properties of a monoclonal DNP/H-2Kd-specific suppressor molecule produced by a Ts hybridoma. Reduction followed by alkylation abrogated the ability of the 3-10 molecule to inhibit transfer of contact sensitivity to 2,4-dinitro-fluorobenzene, indicating that intact disulfide bonds were a required structural property for suppression. Reduction of the 3-10 molecule followed by affinity chromatography on DNP-coupled Sepharose beads indicated that the 3-10 suppressor molecule is a dimer and that one of its chains binds to cell-free DNP. Serologic properties of the 3-10 molecule were examined by determining the ability of pan-reactive rabbit anti-TCR antibodies and anti-V beta 8 mAb KJ16.133 and F23.1 to adsorb suppressor activity from 3-10 culture supernatant and affinity purified 3-10 ascites material. All three reagents adsorbed the suppressor activity whereas control antibodies had no effect. When 3-10 material was passed through a F23.1-conjugated Sepharose affinity column, suppressor activity was recovered in the column eluate but not in the effluent fraction. When the 3-10 molecule was reduced and separated into its two chains (i.e., DNP-binding and non-DNP-binding chains), it was found that the anti-V beta 8 antibody F23.1-bound to the non-DNP-binding chain of the suppressor molecule. Collectively, these results indicate that the monoclonal 3-10 suppressor molecule is structurally similar to the alpha/beta TCR and suggest that the 3-10 molecule expresses a determinant encoded by the V beta 8 family of TCR genes. These results are consistent with our hypothesis that these suppressor molecules represent a secreted form of the TCR expressed on the surface of the DNP-specific Ts.

Animals↗
PubMed1988

Epitopes associated with major histocompatibility complex (MHC) restriction site of T cells. IV. I-J epitopes on MHC-restricted cloned T cells.

We studied the expression of an I-Jk epitope on class II-restricted cloned L3T4+ T cells established from H-2k, H-2b, F1 and semiallogeneic radiation bone marrow chimeras by the inhibition of antigen-induced T cell proliferation and in vitro secondary antibody response, and by the direct immunofluorescence with a monoclonal anti-I-Jk. Both I-Ak- and I-Ek-restricted T cells were shown to carry the identical I-Jk epitope regardless of their genotypic origins, antigen specificity, and helper or suppressor function. None of the I-Ab-restricted clones derived from similar animals showed the I-Jk epitope. This isomorphism, regardless of the restriction specificity for I-Ak or I-Ek, contradicts the idea that I-J is an idiotypic determinant on class II-restricted T cell antigen receptor (TcR). In fact, the I-Jk epitope was not comodulated with TcR/T3 complex when incubated with an anti-T3 antibody, indicating that I-J is a new isomorphic receptor for self different from TcR alpha/beta heterodimers.

Animals↗
PubMed1987

Generation and use of an antigen-specific hybrid to study B-cell function.

Several important events are known to occur following the crosslinking of membrane-associated immunoglobulin (mIg) on B-lymphocytes. Among these include the internalization and re-expression of mIg. We have addressed two questions regarding the re-expression of mIg following an antigen-induced clearance of this B-cell receptor. 1) How long does it take for a cell to re-express its receptors after the first or second exposure to antigen, and 2) is this re-expression dependent upon the synthesis of new mIg? These questions, which in the past have been addressed primarily with heterogeneous populations of cells and using anti-immunoglobulin instead of antigen, are vital to understanding B-cell activation as well as antigen processing events. In order to address these questions, we have produced and characterized an antigen-specific B-cell hybrid 2C3E1 that expresses a membrane-associated form of immunoglobulin. A hybrid specific for a charged hapten (phthalate) was selected to avoid the possibility of nonspecific hydrophobic interactions of the binding ligand with the plasma membrane. After establishing the presence of mIg biochemically and demonstrating the ability of phthalate-keyhole limpet hemocyanin to induce the clearance of the phthalate-specific receptor from the plasma membrane, it was determined that re-expression of antigen-specific receptors was detected within one hour and completed by six hours after the first or second pulse with antigen and that this re-expression did not require the synthesis of new receptors. This later finding is novel and suggests that B-cell receptors are re-utilized or that a presynthesized pool of mIg exists within the cytoplasm of these cells. During the characterization of the 2C3E1 cell line, it was determined that this hybrid also produced a secreted form of the phthalate-specific immunoglobulin (sIg). An unexpected subsequent observation was that sIg was found associated with the cell surface in addition to membrane Ig. A likely source of this unexpected surface-associated sIg was observed as a vesicle on the surface of many cells that is associated with a high concentration of sIg. This finding and its possible relevance to Ig secretion per se are considered further in the paper which follows this one.

Animals↗
PubMed1987

Biochemical and ultrastructural characterization of a novel cell structure associated with immunoglobulin secretion in B-lymphocytes.

In the companion paper, it was established that a secretory form of immunoglobulin, sIg, is present at or near the cell surface. This unexpected occurrence of sIg was postulated to be due to the labelling of sIg which remains temporarily associated with the cell packaged in a vesicle which appears to bud from the plasma membrane at a single pole of the cell. The question that is addressed in this report is whether or not this polar accumulation of sIg represents a common pathway for the exit of this protein which is used by antibody-producing cells. This question is important since, in spite of the fact that the intracellular events associated with immunoglobulin synthesis (processing and movement between subcellular compartments) have been defined, very little data exists on how or where immunoglobulin finally leaves the plasma cell. This question was approached here by first demonstrating that the polar immunoglobulin secretory vesicles (ISV) are associated with several sIg-producing cells including other hybridomas, B-cell lines, and mitogen-activated spleen cells. The second approach was to characterize the ISV on the cell ultrastructurally and to establish that these vesicles are released from the cell carrying with them sIg. Isolated vesicles released from biosynthetically labeled Ig-producing cells were analyzed by SDS-PAGE in order to confirm the presence of sIg and to determine the number of other proteins associated with the ISV, their molecular weights, and the degree of disulfide crosslinking of the molecules comprising this structure. Finally, the kinetics of sIg release was established by a pulse chase protocol for biosynthetically labeled cells, and by monitoring the release of radioactive Ig from surface iodinated cells. As was predicted from our biochemical studies of the ISV, we observed a very slow phase of sIg release as well as a rapid release phase. Our studies have established that at least one of the pathways for the release of Ig from hybridomas, B-cell lines, and normal splenic B-cells is via a polar multivesiculated structure that we have termed ISV, and that the sIg can be released either as a free form of the protein or packaged within a satellite vesicle which may release the sIg later and perhaps at considerable distance from the cell that produced it.

Animals↗
PubMed1986

Involvement of the T cell antigen receptor and of Lyt-2 in the cytotoxic function of aged killer (AK) T cells.

Aged killer (AK) T cells are antigen-independent, IL-2-requiring variants of antigen-dependent CTL clones that have lost their original antigen specificity and have acquired, instead, specific cytotoxicity for P815 target cells. In this report we study whether AK cells use a similar or a different target cell recognition system than that of bona fide CTL. To this end, we selected from a cloned AK line variants that are partially or completely deficient in specific target recognition and/or in cytotoxic function, and analyzed these variants for expression of the T cell antigen receptor and of Lyt-2. Variants were selected from the prototype AK line (Cl 96) with specific, as well as lectin-facilitated, cytotoxicity for P815 tumor cells. Variants could be grouped into four types with increasing degrees of functional deficiency, which correlated with loss of T cell receptor and/or loss of Lyt-2. In short, loss of Lyt-2 was reflected in loss of specific target recognition, and loss of the T cell antigen receptor was reflected in loss of all cytotoxic activity. We conclude from these results that both Lyt-2 and the T cell antigen receptor are required for specific target cell recognition and the T cell antigen receptor is, in addition, required for cytotoxic function. Moreover, since AK cells express a somatically acquired specificity that differs from that of their clonal precursors, it appears that cytotoxic T cells may change their antigen receptor from one specificity to another during tissue culture.

Animals↗
PubMed1986

Ia-specific mixed leukocyte reactive T cell hybridomas: analysis of their specificity by using purified class II MHC molecules in synthetic membrane system.

This study was undertaken to determine the nature of the antigens recognized in allogeneic and syngeneic mixed leukocyte reactions (MLR). Specifically, we wished to determine whether Ia antigens alone were recognized by MLR-reactive T cells, or whether the specificity was determined by the corecognition of non-MHC antigens together with syngeneic or allogeneic Ia. To do this we used 11 T cell hybrids that were characterized as being specific for Iad and were tested their capacity to respond to isolated I-Ad or I-Ed that had been incorporated into liposomes and had bound to the surface of glass beads. Of nine alloreactive T cell hybrids (five I-Ad-and four I-Ed-specific), seven were shown to be responsive to the relevant isolated Ia antigen on glass beads. Also, two of two syngeneic I-Ad-specific T cell hybrids responded to I-Ad on the glass beads. One of the two alloreactive T cell hybrids that failed to respond to the relevant Ia antigen on glass beads was shown to be specific for an antigen in fetal calf serum (FCS) that was recognized in the context of the allo-Ia antigen (I-Ed), because when intact accessory cells were used, a response by this hybrid was only observed when FCS was present in the assay culture medium or when the accessory cells were pre-pulsed with FCS. The possible involvement of FCS antigens and non-Ia accessory cell antigens in the stimulation of the nine T cell hybrids that responded to isolated Ia on glass beads was evaluated. T cell hybrids that were grown and were tested in serum free medium were still capable of reacting to Ia on beads. The isolated Ia preparations used were greater than 90% pure, and their capacity to stimulate the T cell hybrids did not correlate with the degree of contamination with non-Ia proteins. We conclude from these studies that the majority of T cells that respond to allogeneic or syngeneic Ia bearing stimulator cells are specific for the Ia antigens themselves, and do not require the co-recognition of other non-Ia antigens; nor is there any requirement for Ia antigen processing for this recognition.

Animals↗
PubMed1986

Preparation and characterization of a "pan-reactive" rabbit anti-mouse T-cell receptor antiserum.

A pan-reactive xenoantiserum to the mouse T-cell receptor was prepared by immunization of a rabbit with affinity purified mouse T-cell receptor material. The T-cell receptor of the chicken ovalbumin/IAd specific T-cell hybridoma, DO-11.10, was isolated by affinity chromatography using the clone-specific monoclonal antibody, KJ1-26. Immunoprecipitation with the rabbit antiserum and subsequent SDS-PAGE analysis of the material precipitated from lysates of surface radioiodinated T cells revealed the heterodimeric structure characteristic of the T-cell receptor from virtually every T-cell source examined. Flow cytofluorometric analysis of normal peripheral T cells and mature thymocytes of BALB/c and SJL mice indicated that most all T cells bear antigenic determinants recognized by the rabbit anti-mouse T-cell receptor antibodies. The AKR thymoma, BW5147, a common fusion parent used to generate functional T-cell hybridomas, notably lacks surface expression of a T-cell receptor molecule.

Animals↗
PubMed1983

Expression of membrane IGM by a human B lymphoblastoid cell line in the presence of monensin.

The effect of the carboxylic ionophore, monensin, on the synthesis and expression of membrane IgM in the human lymphoblastoid cell line, Daudi, was investigated. The normal processing events in the maturation of mu chains and k chains were altered in monensin treated Daudi cells; the immunoglobulin chains did not appear to undergo complete terminal glycosylation modifications. Transport of the glycoprotein to the plasma membrane could be demonstrated indicating that the interference of intracellular processing of the IgM by monensin did not significantly influence the membrane expression of the IgM.

B-Lymphocytes↗
PubMed1983

Tunicamycin inhibits the expression of membrane IgM in the human lymphoblastoid cell line Daudi.

Tunicamycin inhibited the synthesis of glycosylated mu-chains and kappa-chains in the human lymphoblastoid cell line, Daudi. Nonglycosylated IgM could not be detected on the surface of tunicamycin-treated cells by cell surface iodination techniques even under conditions where membrane IgM was re-expressed in control cultures following the enzymatic stripping of the existing membrane IgM. Biosynthetic labeling and subsequent immunochemical analysis indicated that the nonglycosylated mu and kappa-chains failed to efficiently assemble into monomeric IgM units. In a previous study (Dulis et al., J. biol. Chem., 1982), we have shown that the nonglycosylated mu- and kappa-chains are rapidly catabolized. The lack of expression of nonglycosylated IgM could be due to the rapid catabolism of the nonglycosylated polypeptide chains and/or to the inability to form functional monomeric IgM molecules. Thus glycosylation may be required to protect the newly synthesized polypeptide chains from intracellular catabolic events and to maintain proper conformational foldings of the polypeptide chains to allow for the assembly of subunits into functional units and their ultimate expression.

Cell Line↗
PubMed1982

Regulation of catabolism of IgM heavy chains in a B lymphoma cell line.

The human lymphoma cell line Daudi has the phenotype of a nonsecreting B cell. This cell line synthesizes both the membrane and secreted forms of the IgM heavy chain, but only expresses functional membrane IgM. We have found that secreted type heavy chains (mus) are rapidly degraded in these cells, with a half-life of 1.3 h. Some of the membrane type heavy chains (mum) are also rapidly catabolized but some are expressed in a stable form with a half-life of 13 h. Inhibiting the initial glycosylation of heavy chains with tunicamycin has differential effects on the catabolic rates of mus and mum chains. The turnover of mus chains is not affected by this inhibitor, but the degradation of mum chains is much more rapid after tunicamycin treatment. In comparison with their glycosylated counterparts, nonglycosylated mu chains do not covalently assemble to a significant degree with light chains. Tunicamycin treatment of Daudi cells thus seems to inhibit formation of stable mum protein, possibly by altering mu chain conformation and inhibiting its interaction with light chains. We conclude from these results that some mum chains are specifically protected from proteolysis by post-translational events. These processing events include covalent assembly with light chains, terminal glycosylation, and insertion into the plasma membrane.

B-Lymphocytes↗
PubMed1979

Studies on the cytophilic properties of human beta2-microglobulin. II. The role of histocompatibility antigens.

The mechanism by which exogenously added beta2m binds to lymphoid cells has been explored. In the mouse it has been shown that beta2m remains associated with plasma membrane macromolecules following solubilization with NP-40 and that approximately 25-30% of the binding could be accounted for by binding to H-2 antigens. No binding to mouse immunoglobulin or Ia antigens could be detected. The sites for binding of the remainder of the cell-bound beta2m were not determined. Whereas normal human lymphocytes showed little or no capacity to bind exogenously added beta2m, it was found that phytohaemagglutinin (PHA)-stimulated cells could bind beta2m. This binding occurred optimally 2 days after PHA stimulation. Approximately half of the binding could be accounted for by binding to HLA antigens. The possible significance of these findings with respect to cellular interactions involving major histocompatibility complex gene products in the immune response is discussed.

Beta-Globulins↗