bluuebunny/crossref_metadata_2025 huggingface dataset:
Activation of human cytolytic cells through CD2/T11. Comparison of the requirements for the induction and direction of lysis of tumor targets by T cells and NK cells.
Authors: Scott C F , Bolender S , McIntyre G D , Holldack J , Lambert J M , Venkatesh Y P , Morimoto C , Ritz J , Schlossman S F
Published: December 2022

We studied the mechanisms whereby human T cells and NK cells are activated and directed to lyse tumor targets through the CD2 (T11/E-rosette) Ag. Using two cloned NK lines, we showed that these cells, as had previously been shown for T cells, could be directed to lyse an "NK-resistant" tumor target in the presence of antibody heterodimers. These heterodimers consisted of a (mAb) to CD2 (anti-T11(2) or anti-T11(3] linked to a mAb recognizing the tumor cell (J5, anti-CALLA). However, distinct differences between NK cells and T cells were observed with regard to the requirements for such directed lysis: first, only one epitope of CD2 on NK cells (either T11(2) or T11(3] needed to be recognized by the antibody heterodimer in order for directed lysis to occur, whereas for T cells both T11(2) and T11(3) epitopes had to be recognized. Second, in confirmation of previous data with monomeric anti-T11(2) or anti-T11(3) antibody, heterodimers constructed with these reagents enhanced conjugate formation between NK cells and tumor targets, whereas no such enhancement was seen with T cells. All types of heterodimer directed lysis were dependent on the adhesion molecule LFA-1, as an anti-LFA-1 antibody-blocked lysis. Third, whereas in T cells lysis mediated through CD2 appeared to be regulated by CD3 but not vice versa, all types of lysis by NK cells appeared to be regulated through CD2. Finally we showed that F(ab')2 fragments of the anti-T11(2) and anti-T11(3) antibodies could activate NK cells, but were unable to activate T cells either as cloned cytolytic lines, or in populations of PBL. The implications of our findings with regard to the role of CD2 in the activation of cytolytic cells is discussed.
Regulation of MHC expression in vivo. II. IFN-alpha/beta inducers and recombinant IFN-alpha modulate MHC antigen expression in mouse tissues.
Authors: Halloran P F , Urmson J , Van der Meide P H , Autenried P
Published: December 2022

We examined the effect of type I IFN inducers and rIFN-alpha on MHC expression in mouse tissues in vivo. MHC expression was assessed in a radiolabeled mAb binding assay and by indirect immunoperoxidase staining of tissue sections. polyI:C, an inducer of IFN-alpha/beta, induced large increases in class I MHC in many tissues, with little effect on class II expression. In the kidney, which was studied in detail, polyI:C increased class I expression from day 1 to day 6, localized in glomeruli, tubules, and arterial endothelium. Renal class II MHC was less affected but tended to be decreased at days 3 to 6, corresponding to diminished staining of class II-positive interstitial cells. polyI:C increased renal class I MHC in nude mice and mice with severe combined immunodeficiency, and in mice treated with cyclosporine or mAb against IFN-gamma. The effects of influenza virus resembled those of polyI:C. However, a potent T cell stimulus, allogeneic ascites tumor cells, induced markedly different MHC changes, with massive and sustained increases in class I and II, presumably due to IFN-gamma release, which was inhibited by cyclosporine or by mAb against IFN-gamma. The effect of polyI:C was largely simulated by rIFN-alpha, whereas the effect of allogeneic cells was simulated by rIFN-gamma. Thus, rIFN-alpha and its inducers in vivo produce a sustained increase in renal class I expression in kidney and other tissues, sometimes with changes in class II expression. Such effects could be relevant to the immune modulatory actions of IFN, and to the immunologic consequences of viral infections.
IL-6 is an intermediate in IL-1-induced thymocyte proliferation.
Authors: Helle M , Boeije L , Aarden L A
Published: December 2022

Both IL-1 and IL-6 have been shown to be comitogenic for lectin-stimulated thymocytes. Thymocytes cultured in the presence of IL-1 produce IL-6 themselves. This IL-6 production is caused by a cell population with low buoyant density. After removal of these cells, IL-6 or IL-2 are still co-mitogenic for thymocytes whereas IL-1 is not. Addition of IL-1 to such thymocytes renders them about 100-fold more sensitive to IL-6. At all conditions proliferation is inhibitable with antibodies to IL-2 and to the IL-2R. Our experiments show that IL-1-driven proliferation of thymocytes is dependent on endogenous IL-6 production and that in the classical thymocyte assay IL-1 has a dual role: it induces IL-6 production and it greatly increases the sensitivity for IL-6.
Arachidonic acid mobilization and phosphoinositide turnover by the terminal complement complex, C5b-9, in rat oligodendrocyte x C6 glioma cell hybrids.
Authors: Shirazi Y , McMorris F A , Shin M L
Published: December 2022

Previously, we have shown that rat oligodendrocytes release phospholipid and generate arachidonic acid (AA) and leukotriene B4 in response to sublytic C5b-9 formation. In the present study, we investigated the biochemical pathways by which C5b-9 generates AA from clone ROC-1, a fusion product of rat oligodendrocytes and C6 glioma. Cells were incubated for 24 h in the presence of [3H]AA or [3H]myoinositol. They were then sensitized with antibody against hybrid cell stroma and treated for 1 h with C9-depleted human serum (C9D-HS) or C9D-HS reconstituted with C9. Alternatively, cells were treated with C8,C9D-HS or C8,C9D-HS reconstituted with C8 or C8 plus C9 for 1 h. Qualitative and quantitative analysis of the released [3H]AA and [3H]myoinositol radiolabeled products were performed by thin layer chromatography/autoradiography and anion exchange chromatography, respectively. The major [3H]AA radiolabeled products after C5b-9 stimulation comigrated with intact phospholipid and AA standards, and the major [3H]myoinositol radiolabeled product was inositol-1-phosphate. Treatment of cells with phospholipase A2 inhibitors, mepacrine and bromophenacyl bromide, abolished AA release by C5b-9. In the absence of extracellular Ca2+, C5b-9 also failed to induce the release of AA. Interestingly, 1-(5-isoquinolinsulfonyl)-2-methylpiperazine (H-7), a potent inhibitor of protein kinases, inhibited AA release by C5b-9, whereas AA release stimulated by the calcium ionophore A23187 was not blocked by H-7. The results suggest that AA generation by C5b-9 from the ROC-1 clone involves activation of Ca2+-dependent phospholipase A2 which is regulated by protein kinase-dependent mechanisms.
Analysis of VH gene replacement events in a B cell lymphoma.
Authors: Kleinfield R W , Weigert M G
Published: December 2022

We have analyzed a series of recombinational events at the IgH chain locus of the B cell lymphoma, NFS-5. Each of these recombinational events results in the replacement of the VH gene segment of the rearranged H chain gene (VhDJh) with that of an upstream germline gene segment. Replacements on the productive and nonproductive alleles have been observed. In each case, the recombination occurs in close proximity to a highly conserved heptameric sequence (5'TACTGTG3') which is located at the 3' end of the VH coding region. In the two examples of recombination on the productive allele that have been analyzed, the initial VHQ52 gene is replaced by different VH7183 genes. On the non-productive allele, sequential replacement events have been analyzed: the initial VHQ52 rearrangement is first replaced by a closely related VHQ52 gene, followed by a second replacement using a VHQ52 pseudogene. Southern blot analysis using VH probes indicates that these recombinations may be accompanied by the deletion of germline VH genes belonging to both the VHQ52 and VH7183 families, suggesting that these gene families are interspersed in the NFS/N mouse.
Generation of T cell repertoire. Two distinct mechanisms for generation of T suppressor cells, T helper cells, and T augmenting cells.
Authors: Asano Y , Tada T
Published: December 2022

The present studies were carried out to characterize the influence of the T cell maturation environment on a repertoire of Th cells, T augmenting cells, and Ts cells, which were shown to construct a minimal regulatory circuit to regulate a helper function of class II-restricted Th cells. A repertoire of keyhole limpet hemocyanin-specific Th cells was influenced predominantly by I-A molecules of the T cell maturation environment, whereas a repertoire of T augmenting cells was determined by both I-A and I-E molecules. These repertoires were not influenced by the priming with Ag. A repertoire of Ts cell factor-producing Ts cells was not influenced by the T cell maturation environment, but rather was determined by the I-J haplotype of the APC utilized for priming with Ag. In contrast, a repertoire of novel cognate type Ts cells, which inhibit class II-restricted Th cell function in a restriction-restricted manner, was influenced by both I-A and I-E molecules of the T cell maturation environment. These results demonstrate the two distinct mechanisms for generating a T cell repertoire: a selection by class II molecules of the T cell maturation environment before the priming with foreign Ag and a selection by priming with APC and Ag.
Generation of somatic variants of a B cell hybrid mediated by a non-cytolytic L3T4+ idiotype-specific T cell.
Authors: Ghosh S K , Bankert R B
Published: December 2022

We previously reported that idiotype (Id)-loss, stable somatic variants of a B cell hybrid, 2C3E1, are generated both in vitro and in vivo, after interaction of the Id-positive tumor cells with autologous Id-specific effector T cells. The present investigation was undertaken to elucidate further the nature and functional characteristics of the effector T cells. We report here that the idiotype-specific cells mediating the generation of Id- tumor variants are Thy1+ L3T4+ Lyt-2- cells, which respond to specific idiotypic stimulation by secreting IL-2 in vitro. No IL-2 is secreted in response to unrelated Ig or an Id/Ig-2C3E1 tumor variant. Furthermore, the Id-specific T cells exert strong suppressive effects on the expression of 2C3E1 Ig and the effects can be reversed by blocking the L3T4+ T cells with monoclonal anti-L3T4 antibody in vitro during the initial 3 days of co-culture. After 4 days, the T cell-mediated suppression of the 2C3E1-Id is irreversible. In addition to the in vitro studies we have determined that the administration of anti-L3T4 mAb to mice just before priming with idiotype-bearing tumor cells also abrogates the suppressive effects of the idiotype primed spleen cells on Ig expression of 2C3E1. To study the Id-specific effector T cells in more detail we have generated functional Id-specific L3T4+ T cell lines. These T cell lines have been shown to recapitulate the generation of Id- tumor variants that we observed with Id-primed spleen cells. It is concluded that L3T4+, Id-specific Ts cells are responsible for the generation of somatic variants of the B cell hybrid 2C3E1 and that the induction or selection of these variants progresses from a reversible phase to an irreversible phase.
The requirements for triggering of lysis by cytolytic T lymphocyte clones. II. Cyclosporin A inhibits TCR-mediated exocytosis by only selectively inhibits TCR-mediated lytic activity by cloned CTL.
Authors: Lancki D W , Kaper B P , Fitch F W
Published: December 2022

TCR-mediated granule exocytosis, as measured by the release of serine esterase activity, has been implicated in the lytic process of Ag-specific CTL. Exocytosis appears to be the mechanism of release of other lysis-relevant molecules including cytotoxic lymphokines and proteins that have the capacity to induce membrane lesions as measured by the hemolysis of non-nucleated SRBC. In the studies presented here, we assessed the contribution of exocytosis and lymphokine production in CTL lysis of nucleated and non-nucleated target cells by using a panel of murine CTL clones. Ag-mediated activation of cytolysis, lymphokine production, and exocytosis could be mimicked by mAb against the TCR/CD3 complex, or by stimulation with the combination of PMA + calcium ionophore, which appear to bypass the TCR (neither PMA nor calcium ionophore alone induced these functions efficiently in our CD8+ CTL clones). Although lysis, IFN-gamma production and exocytosis of N-alpha-benzyloxycarbonyl-L-lysin esterase (BLTE) activity were induced by either stimulus, we were able to identify distinct activation requirements for each of these functions. We found that lymphokine production, exocytosis, and cytolysis could be selectively inhibited. Cycloheximide inhibited IFN-gamma production, but did not inhibit exocytosis of BLTE activity or cytolysis. In addition we showed that cyclosporine A (CsA) profoundly inhibited IFN-gamma production as well as exocytosis induced by stimulation through the Ag receptor or by PMA + calcium ionophore. In contrast, CsA had little or no effect on lysis of nucleated target cells that bear the relevant Ag. These findings indicate that our CTL clones can lyse target cells by a mechanism independent of exocytosis or (de novo) lymphokine production. To directly assess the capacity of our CTL clones to lyse target cells without inducing nuclear damage we developed a system of coating non-nucleated SRBC with anti-CD3 mAb for use as stimuli and as targets for lysis. We found that our cloned CTL were indeed activated to produce IFN-gamma by SRBC that were coated with anti-CD3 mAb, and, furthermore, they were able to lyse the SRBC in a short term cytolytic assay. Thus our CD8+ CTL are capable of lysing certain target cells by a mechanism independent of DNA degradation, presumably by inducing a membrane lesion. In addition, CsA did inhibit lysis of the non-nucleated SRBC targets as well as exocytosis of BLTE activity.(ABSTRACT TRUNCATED AT 400 WORDS)
Two monoclonal antibodies identify thymic-repopulating cells in mouse bone marrow.
Authors: Spangrude G J , Klein J , Heimfeld S , Aihara Y , Weissman I L
Published: December 2022

The progenitor cells in the bone marrow that home to and repopulate the thymus have been incompletely characterized. In particular, it is not clear whether thymocytes differentiate directly from pluripotent hemopoietic stem cells that seed to the thymus, or whether T lymphoid-committed stem cells (prothymocytes) arise in the bone marrow before the thymic migration. In order to resolve this question, we have used mAb specific for cell-surface Ag to identify the bone marrow cells which can seed to and repopulate the thymus of irradiated mice. We report here that the majority of thymic-repopulating cells in mouse bone marrow express low levels of the Thy-1 Ag (Thy-1lo) plus high levels of a newly described Ag termed stem cell Ag (Sca-1). Two distinct populations of thymic-repopulating Thy-1loSca-1+ cells in mouse bone marrow can be discriminated based on expression of any of a number of hemolymphoid lineage-specific (Lin) markers. Thus, Thy-1loLin-Sca-1+ and Thy-1loLin+Sca-1+ fractions of bone marrow contain thymic-repopulating cells. A second Ag, stem cell Ag-2 (Sca-2), is expressed by Thy-1loLin+Sca-1+ cells but not by Thy-1loLin-Sca-1+ cells. The Thy-1loLin-Sca-1+ fraction expresses intermediate levels of the phagocyte glycoprotein-1 Ag, and comprises 30% of the Thy-1loLin- bone marrow cells, which have previously been shown to be highly enriched in pluripotent hemopoietic stem cells. By facilitating the isolation of highly purified subpopulations of bone marrow cells that can repopulate the thymus, Sca-1 and Sca-2 should provide an experimental tool for describing the developmental potential of such bone marrow subsets.
A natural neonatal hybridoma autoantibody to the T200 antigen.
Authors: Glotz D , Zanetti M
Published: December 2022

We report on the production and characterization of a murine hybridoma generated from neonatal (less than 24 h from birth) unstimulated BALB/c splenocytes that produces an IgG2b kappa-antibody (21G10) reacting with a cell surface Ag of murine lymphocytes. By immunoprecipitation technique we determined that the Ag recognized by autoantibody 21G10 has an apparent m.w. of 200 kDa and is present on both T and B lymphocytes but not on fibroblasts. Together with the pattern of immunoprecipitation and the cell lineage distribution we suggest that autoantibody 21G10 likely recognizes the common leukocyte Ag T200. This is further inferred by using a mutant (T200-) cell line and a reference anti-T200 mAb in immunodepletion experiments. Functionally, autoantibody 21G10 blocks (greater than 90%) the incorporation of [3H]TdR by T lymphocytes stimulated in vitro with Con A, and inhibits the production of IL-2 by these cultures. This report demonstrates the existence of autoantibodies directed against lymphocyte cell surface Ag within the natural preimmune repertoire. The implication of this finding with regard to T-B cells interaction early in ontogeny is discussed.
The xid mutation affects hemopoiesis in long term cultures of murine bone marrow.
Authors: Hayashi S , Witte P L , Kincade P W
Published: December 2022

Cells of the humoral immune system are particularly affected by a mutation at the X chromosome linked immunodeficiency disease (xid)locus. Although B cells are made in normal numbers, they fail to become phenotypically and functionally diverse. Consequently, poor antibody responses are mounted to certain types of Ag. There have been some indications that other types of hemopoietic cells may be influenced by the mutation and development of the humoral immune system is unusually dependent on the presence of T lymphocytes. We now describe an analysis of the lympho-hemopoietic environment studied with long term bone marrow cultures. Contrary to expectations, cultures initiated with cells from homozygous female or hemizygous male mice with the mutant allele established more quickly than normal. The accelerated initial growth pattern was clearly linked to the xid mutation. Artificial mixtures of marrow exhibited intermediate growth kinetics. Experiments with H-2 congenic and T6 chromosome marked cells did not reveal an intrinsic dominance of growth in nonadherent xid cells. Similar results were obtained with culture conditions which favored production of myeloid or lymphoid cells. These findings would be consistent with subtle changes in the bone marrow microenvironment resulting from the xid mutation. The pedigree of the mouse strains had a significant influence on lymphopoiesis in long term bone marrow cultures. Lymphocytes of BALB/c origin dominated over CBA/H background cells in cultures established from mixtures of the two, but this did not correlate with any functional deficiency in CBA/H stromal cells. In fact, establishment of an adherent layer was a rate-limiting step in initiating long term cultures and this could be achieved with a low dose inoculum of CBA/H marrow. Even more dramatic effects were found in hemopoietic cells from doubly defective C3H.nu/nu-xid mice. The bone marrow of these athymic animals contained normal numbers of granulocyte/macrophage progenitors. However, lymphoid cultures could not be reproducibly established with their cells and myelopoiesis was never observed in vitro. The relatively simple conditions which pertain in culture make it possible to appreciate effects of mutations and pedigree on hemopoiesis which are unremarkable in intact animals.
Regulation of monocyte/macrophage C2 production and HLA-DR expression by IL-4 (BSF-1) and IFN-gamma.
Authors: Littman B H , Dastvan F F , Carlson P L , Sanders K M
Published: December 2022

IL-4 was originally described on the basis of its ability to co-stimulate the proliferation of resting B cells treated with anti-IgM. Recently, this cytokine has been shown to have other effects on mast cells, T cells, B cells, and macrophages. We studied the ability of IL-4 to regulate the production of C2 by human monocytes and monocytic cell lines and compared this with stimulation of HLA-DR expression, another recently described activity of IL-4. Responses to IL-4 were compared to IFN-gamma, a cytokine with both activities. IL-4 up-regulated C2 production by human monocytes and this effect was not inhibited by neutralizing anti-IFN-gamma antibody. IL-4 also stimulated C2 production by HL-60 cells that had been pre-treated with vitamin D3 to induce monocytic differentiation. IL-4 did not stimulate C2 production by U937 cells. IFN-gamma, in contrast to IL-4, stimulates C2 production by all three cell types. Although IL-4 increased C2 production by HL-60 cells we could not detect C2 mRNA by Northern blotting. However, co-stimulation of these cells with IL-4 and low concentrations of IFN-gamma resulted in an additive effect on C2 production and a greater increase in C2 mRNA than was seen with IFN-gamma alone. As reported by others, IL-4-stimulated HLA-DR expression by monocytes. In contrast to our findings regarding C2 production, stimulation of HLA-DR expression was inhibited by neutralizing anti-IFN-gamma mAb and IL-4 did not stimulate HLA-DR expression by U937 or HL-60 cells. IFN-gamma stimulated HLA-DR expression by all three cell types. These results identify IL-4 as an additional cytokine able to directly stimulate C2 production by human monocytes and by a monocytic cell line whereas IL-4 stimulation of HLA-DR expression by monocytes appears to be IFN-gamma dependent.
Two steps in the intracellular transport of IgD are sensitive to energy depletion.
Authors: Argon Y , Burkhardt J K , Leeds J M , Milstein C
Published: December 2022

The secretory pathway of murine IgD can be dissected by the use of carbonylcyanide m-chlorophenylhydrazone (CCCP), which inhibits two distinct steps of intracellular transport. The newly synthesized IgD that accumulates at the first step contains high mannose type oligosaccharides which are partially trimmed. The IgD arrested at this step is less processed than the IgD arrested by treatment with monensin. The properties of this biosynthetic intermediate are consistent with inhibition of Ig passage from the endoplasmic reticulum to the Golgi complex. A second CCCP-sensitive step exists in the biosynthesis of IgD, and is characterized by delta-chains that are resistant to endoglycosidase H and contain galactose. This indicates that this second step occurs during or after the passage through the trans-Golgi compartment. The galactose-containing oligosaccharides of the delta-chains arrested at this step do not contain fucose (as do mature, secreted delta-chains). Fucosylation is not inhibited by CCCP, nor is the secretion of fucose-containing delta-chains. These results show that terminal sugars are added to secretory IgD in at least two transport compartments, separable by their sensitivity to CCCP. The inhibition of the secretory pathway at both steps is reversible; upon removal of the drug the arrested IgD is processed normally and is secreted. The sensitivity to CCCP probably reflects transport steps that are sensitive to even partial depletion of ATP, because treatments with other inhibitors of oxidative phosphorylation yield similarly arrested Ig molecules. Thus, by using the protonophore CCCP, we demonstrate two energy-requiring steps in IgD transport which seem to be at two transitions in the secretory pathway. One step is during the passage from the endoplasmic reticulum to the mid-Golgi compartment and the other step is during Ig passage through the trans-Golgi, or subsequent transport to the cell surface.
Biochemical characteristics and partial amino acid sequence of the receptor-like human B cell and carcinoma antigen CDw40.
Authors: Braesch-Andersen S , Paulie S , Koho H , Nika H , Aspenström P , Perlmann P
Published: December 2022

The Ag CDw40 (p50, Bp50) is a phosphoprotein expressed on the surface of both B lymphocytes and on certain malignant cell types of nonhemopoietic origin. Antibodies to this Ag have been shown to act as a potent co-mitogen for B cells. In order to elucidate the function of this Ag, we have now investigated some of its biochemical characteristics as well as the relationship of B cell derived CDw40 to that derived from urinary bladder carcinoma (transitional cell carcinoma, TCC) cells. CDw40 from normal B cells or from the Burkitt lymphoma line Raji showed a characteristic pattern of three bands when analyzed by SDS-PAGE and Western blotting: a main band of 47 kDa, a degradation product of 43 kDa, and a dimer of 85 kDa. The dimer was disrupted by reduction with 2-ME but was reformed spontaneously from the purified monomers under nonreducing conditions. CDw40 from two bladder cancer cell lines gave a similar pattern but formed little or no dimer. Thirty amino acids of the amino terminal end of CDw40 from Raji and 22 amino acids of that from TCC cells (HU549) were sequenced. The sequences were unusually rich in cysteines and differed only in that the cysteine in position 6 in Raji CDw40 had been replaced by glutamine in HU549. In addition there were two conservative changes in positions 15 and 19. Taken together these results show that CDw40 derived from B cells or from TCC cells are the same or closely related molecules. Comparisons of the amino acid sequence and biochemical characteristics of CDw40 with proteins having receptor functions indicated a close structural resemblance of CDw40 to the nerve growth factor-receptor.
Differential IgG-binding characteristics of staphylococcal protein A, streptococcal protein G, and a chimeric protein AG.
Authors: Eliasson M , Andersson R , Olsson A , Wigzell H , Uhlén M
Published: December 2022

Various Gram-positive bacteria express different types of IgG-binding receptors, each of which displaying certain unique binding properties. To evaluate specificity and avidity aspects of the differential binding pattern, a set of competitive binding assays was employed, by using staphylococcal protein A (SPA), streptococcal protein G (SPG), and a chimeric protein AG. These receptors were analyzed, in a reciprocal fashion, for binding and inhibition of binding to a selected panel of polyclonal and monoclonal Ig. Results of the study reveal that a majority of the determinants on human and bovine IgG, recognized by SPA and SPG, are either coextensive or closely overlapping. Accordingly, a minor portion of the determinants appear to be unique in the sense that a particular determinant(s) is selectively identified by one of the two receptors. Binding assays involving purified Fc fragments from human IgG, suggest that SPG shows exclusive specificity for an Fab region determinant(s) not recognized by SPA, whereas the Fc determinants for SPA and SPG are identical or overlapping. Furthermore, one of the IgG subclasses of bovine origin appears to be seen by the SPG receptor only. The competition study also demonstrates that the novel chimeric protein AG receptor shows higher or equal avidity for variants of human IgG molecules compared to the best of its parental constituents. It can thus be deduced that chimeric receptors might be useful as optimized tools for immunologic applications.
The human B lymphocyte and carcinoma antigen, CDw40, is a phosphoprotein involved in growth signal transduction.
Authors: Paulie S , Rosén A , Ehlin-Henriksson B , Braesch-Andersen S , Jakobson E , Koho H , Perlmann P
Published: December 2022

The human B lymphocyte and carcinoma-associated Ag, CDw40, (p50, Bp50) is a receptor candidate for normal growth regulation. Interaction of mAb with this pan-B Ag, together with preactivating agents such as 12-O-tetradecanoylphorbol-13-acetate or anti-mu, deliver strong growth-promoting signals to the cells. We here demonstrate that signaling through this Ag is dependent on its aggregation on the cell surface. Thus, monovalent antibody fragments were relatively inefficient in this respect but effectively blocked stimulation by intact antibody. By using affinity purified CDw40 protein we have also demonstrated that it is antigenically distinct from other B cell-associated Ag, including the six differentiation clusters CD19 to CD24. The mAb S2C6 and G28.5, prepared by immunizing mice with human bladder carcinoma cells or tonsillar B-cells, respectively, were the only antibodies giving detectable binding. Either of these antibodies could also completely block the binding of the other, suggesting an identity or structural proximity of the epitopes recognized. The CDw40 Ag was shown to be a phosphoprotein lacking intrinsic protein kinase activity. The results provide further evidence for CDw40 being an important B cell growth factor receptor which may also have growth regulatory functions in the development of certain human carcinomas.
Expression of ets genes in mouse thymocyte subsets and T cells.
Authors: Bhat N K , Komschlies K L , Fujiwara S , Fisher R J , Mathieson B J , Gregorio T A , Young H A , Kasik J W , Ozato K , Papas T S
Published: December 2022

The cellular ets genes (ets-1, ets-2, and erg) have been identified by their sequence similarity with the v-ets oncogene of the avian erythroblastosis virus, E26. Products of the ets-2 gene have been detected in a wide range of normal mouse tissues and their expression appears to be associated with cell proliferation in regenerating liver. In contrast, the ets-1 gene was previously shown to be more highly expressed in the mouse thymus than in other tissues. Because the thymic tissue contains various subsets of cells in different stages of proliferation and maturation, we have examined ets gene expression in fetal thymocytes from different stages of development, in isolated subsets of adult thymocytes, and in peripheral T lymphocytes. Expression of the ets-1 gene was first detected at day 18 in fetal thymocytes, corresponding to the first appearance of CD4+ (CD4+, CD8-) thymocytes, and reaches maximal/plateau levels of expression in the thymus at 1 to 2 days after birth. The ets-2 gene expression is detected at least 1 day earlier, coinciding with the presence of both double-positive (CD4+, CD8+) and double-negative (CD4-, CD8-) blast thymocytes and reaches maximal/plateau levels 1 day before birth. In the adult thymus, ets-1 and ets-2 mRNA expression is 10- to 8-fold higher respectively in the CD4+ subset than in the other subsets examined. Higher levels of p55 ets-1 protein were also shown to exist in the CD4+ subset. Because the CD4+ thymic subset is the pool from which the CD4+ peripheral, helper/inducer T cells are derived, the ets gene expression was examined in lymph node T cells. Both the CD4+ and the CD8+ T cells subsets had lower ets RNA levels than the CD4+ thymocytes. These results suggest that ets-2 and more particularly ets-1 gene products play an important role in T cell development and differentiation and are not simply associated with proliferating cells, which are observed at a higher frequency in fetal thymocytes, or dull Ly-1 (low CD5+), and double-negative (CD4-, CD8-) adult thymocytes. Selectively enhanced expression of ets-1 gene may be observed in thymic CD4+ thymocytes because these cells have uniquely encountered MHC class II or other Ag in the thymic environment. These cells may have been subsequently stimulated to activate the ets genes in conjunction with their differentiation of helper/inducer function(s) and expression of mature TCR.(ABSTRACT TRUNCATED AT 400 WORDS)
Characterization of four homologous L chain variable region genes that are related to 6B6.6 idiotype positive human rheumatoid factor L chains.
Authors: Liu M F , Robbins D L , Crowley J J , Sinha S , Kozin F , Kipps T J , Carson D A , Chen P P
Published: December 2022

A significant proportion of monoclonal IgM rheumatoid factors (RF) from patients with mixed cryoglobulinemia express a KIIIa L chain-associated cross-reactive idiotype, termed 6B6.6. Previously, we reported the isolation from a patient with a monoclonal RF of a Vk gene, termed Humkv328, whose deduced amino acid sequence differs by three and seven residues from the L chains of the 6B6.6-positive RF Les and Pom, respectively. To further delineate the genetic basis of the 6B6.6 cross-reactive idiotype, we isolated from the same patient another Vk gene, termed Humkv329, which differs from kv328 by only three bases over a stretch of 1331 nucleotides sequenced. However, kv329 has a stop codon at amino acid position 94, and is thus a pseudogene. Therefore, we screened a second genomic library from an unrelated individual and isolated two potential function Vk genes (i.e., Humkv328h2 and Humkv328h5) which are highly homologous to Humkv328. All three potential functional Vk genes differ from each other by one to six bases over a 1331-bp stretch and all encode the same Vk region amino acid sequence. Among the six bases by which Humkv328h5 differs from Humkv328, two are in the conserved pentadecanucleotide region, which is known to be important in the regulation of k L chain transcription. In the future it will be important to ascertain the potential association of polymorphisms in the conserved pentadecanucleotide region with RF associated autoimmune and lymphoproliferative diseases.
Site-specific mutagenesis of the class I regulatory element the Q10 gene allows expression in non-liver tissues.
Authors: Handy D E , Burke P A , Ozato K , Coligan J E
Published: December 2022

Classical transplantation Ag are found on nearly all cells, whereas Ag encoded by the genes of the Qa/Tla region have a restricted tissue distribution. To investigate the cause of these different patterns of expression, we have compared the regulatory regions of Q10, a Qa region gene that is expressed only in liver, with those of H-2Ld. Gel retardation analysis shows that the nuclear factor (rI) that binds to the inverted repeat (TGGGGATTCCCCA) of the class I regulatory element (CRE) present in H-2Ld and other classical class I genes does not bind to the equivalent region of the Q10 gene. However, the Q10 CRE binds another nuclear factor (rII) that binds to the H-2Ld CRE. The sequence of the Q10 CRE differs from the sequence present in classical class I genes by three nucleotides, two of these changes are within the inverted repeat sequence (TGaGGAcTCCCCA) and disrupt the region of dyad symmetry. We have used site-specific in vitro mutagenesis to individually change these two nucleotides and have investigated the ability of this region to promote transcription with and without these substitutions. A change of either base restores transcriptional activity in chloramphenicol acetyl transferase assays and allows for binding of the rI nuclear factor. These results suggest that the failure of the Q10 CRE to bind the rI nuclear factor may play a role in preventing Q10 expression in tissues other than the liver and fetal yolk sac. Thus, the dichotomy between the widespread tissue expression of classical class I genes and the restricted tissue expression of class I genes of the Qa/Tla region may be due in part to differences in the cis acting regulatory sequences that interact with trans-acting nuclear factors to direct transcription.
Characterization of "regulatory" idiotope-specific T cell clones to a monoclonal anti-idiotypic antibody mimicking a tumor-associated antigen (TAA).
Authors: Saeki Y , Chen J J , Shi L F , Raychaudhuri S , Köhler H
Published: December 2022

As reported previously, 3A4 is a paratope-specific anti-idiotypic mAb and induces cellular and humoral anti-tumor-associated Ag (TAA) responses. In this study, the specificity, MHC restriction and Ag processing requirement of Th cell lines and clones were determined that recognize an idiotypic determinant (Id) on 3A4. The anti-Id-3A4 is part of a tumor-associated idiotypic network which is involved in the regulation of the immunity against the DBA/2 L1210/GZL tumor. These 3A4-Id-specific T cell clones are phenotypically Th cells and recognize Id in the context of MHC class II molecules under MHC restriction. Moreover, the recognition of Id by these T cell clones is chloroquine sensitive, suggesting that they recognize processed Id. However, the 3A4-Id-specific T cell clones respond only to 3A4 and not to TAA. Because these clones do not recognize TAA, their biologic role in antitumor immunity could be as regulatory T cells involved in the idiotypic network regulation.
Induction of tumor-specific in vivo protective immunity by immunization with tumor antigen-pulsed antigen-presenting cells.
Authors: Shimizu J , Suda T , Yoshioka T , Kosugi A , Fujiwara H , Hamaoka T
Published: December 2022

The present study investigates the role of APC in inducing tumor-specific in vivo protective immunity. Thy-1+ cell-depleted, Mac-1+ cell-enriched fraction of normal BALB/c spleen cells were used as a source of APC. These APC were cultured in vitro with the membrane fraction isolated from CSA1M fibrosarcoma derived from BALB/c strain. The administration of such APC into naive BALB/c mice generated the capacity of these animals to reject the subsequently challenged viable CSA1M tumor cells. Although the induction of anti-CSA1M in vivo protective immunity required three consecutive immunizations with more than 10(5) APC which had been pulsed in vitro with 200 to 300 micrograms protein of CSA1M membrane fraction, the immunity was induced irrespective of whether APC were administered via s.c., i.v., or i.p. route. This immunity was tumor-specific, inasmuch as the inoculation of CSA1M or Meth A fibrosarcoma membrane component-pulsed APC resulted in the selective immunity against the challenge with homologous types of tumor cells. The CSA1M-specific in vivo protective immunity was also induced by injecting APC pulsed with solubilized CSA1M membrane components. Moreover, it was demonstrated that the efficiency for inducing anti-CSA1M immunity was much higher in the utilization of tumor Ag-pulsed APC than in the immunization with tumor Ag emulsified in CFA. These results indicate the critical role of APC in generating tumor rejection immunity in vivo and this model presents a novel approach to induce tumor-specific immunity without using tumor cells themselves.
The intracellular precursor of IL-1 beta is associated with microtubules in activated U937 cells.
Authors: Baldari C T , Telford J L
Published: December 2022

Treatment of the human histiocytic leukemia cell line U937 with PMA and LPS results in the induction of expression of IL-1. Rabbit polyclonal antibodies raised against mature rIL-1 have been used to investigate the intracellular location of the IL-1 beta precursor in U937 cells. The pattern of indirect immunofluorescence staining seen with these antibodies overlaps substantially with that seen by using a mAb raised against beta-tubulin. Depolymerization of tubulin by incubation of the cells at 0 degrees C before fixation results in the disruption of the pattern of IL-1 staining. IL-1 beta precursor in extracts of activated U937 cells is shown to co-cycle with exogenously added tubulin through two rounds of in vitro depolymerization/polymerization. In addition, immunoprecipitation of IL-1 from cell extracts at 30 degrees C but not at 0 degrees C results in co-precipitation of tubulin. Thus the IL-1 beta precursor is shown in vivo and in vitro to associate with the microtubules of the cytoskeleton.
IL-6 modulates the synthesis of a specific set of acute phase plasma proteins in vivo.
Authors: Marinkovic S , Jahreis G P , Wong G G , Baumann H
Published: December 2022

Human rIL-6, produced either in COS cells or Escherichia coli, similarly stimulates the production of acute phase plasma proteins in cultured human and rat hepatoma cells. This anabolic effect in hepatoma cells suggested a potential in vivo role of the cytokine in mediating the hepatic response to inflammation. Injection of IL-6 into adult male rats elicited a cytokine-specific change in the liver expression of acute phase proteins. As predicted from in vitro studies, glucocorticoids were needed to achieve a maximal IL-6 response in vivo. Optimal conditions were found to be two i.p. injections of 35 to 120 micrograms IL-6 and 65 micrograms dexamethasone per kg body weight administered at 12-h intervals. Within 24 h, the plasma concentrations for alpha 2-macroglobulin, fibrinogen, thiostatin, and hemopexin were increased to levels approximating those observed in acute phase animals. These results support the notion that direct interaction of IL-6 with the liver is an essential part in initiating the hepatic acute phase reaction.
Prostaglandin E2-dependent induction of granulocyte-macrophage colony-stimulating factor secretion by cloned murine helper T cells.
Authors: Quill H , Gaur A , Phipps R P
Published: December 2022

PG are known to inhibit T cell proliferation, at least in part by suppressing IL-2 production, but effects of PG on the production of other lymphokines have not been well studied. We have found that PGE2 and PGE1, but not PGF2 alpha, inhibit both proliferation and production of granulocyte-macrophage (GM)-CSF by murine TH clones stimulated with Ag or anti-CD3 antibody. Thus, signals generated via the Ag receptor:CD3 complex were inhibited by PGE. Most interesting, however, was the finding that PGE2 and PGE1 could act synergistically with IL-2 for the induction of GM-CSF in some TH1 clones. Dependence on PGE2 for this response was not found in all clones, as some TH1 cells could produce GM-CSF after IL-2 alone, and some cells did not produce GM-CSF even in the presence of PGE2 and IL-2. These observations indicate that there is a subset of TH1 cells receptive to a stimulating activity of PGE2 in the presence of IL-2. PGE2 is known to elevate cAMP levels in T cells. Therefore, we tested whether other agents known to increase cAMP, such as forskolin and cholera toxin, could act in conjunction with IL-2 to induce GM-CSF secretion. As was found with PGE2, these compounds also induced GM-CSF activity in the presence of IL-2, suggesting a critical role for cAMP in this process. Overall these data indicate that the requirements for activation of GM-CSF secretion vary among individual T cells. Most importantly they provide the first evidence that E-series PG are positive signals for lymphokine induction in certain T cells, whereas simultaneously acting as negative signals limiting proliferation. This result also suggests that treatment with anti-inflammatory drugs that decrease PGE2 concentrations may inhibit lymphokine secretion normally stimulated by this pathway.
Human B cell proliferation in response to IL-4 is associated with enhanced production of B cell-derived growth factors.
Authors: Tadmori W , Lee H K , Clark S C , Choi Y S
Published: December 2022

To investigate the capacity of human IL-4 to function as a B cell growth factor (BCGF), we studied its ability to promote proliferation of a selected B cell line. We show that the cell line, designated A4, proliferated in response to IL-4 in a dose-dependent manner. The A4 cells also proliferated in response to their own B cell derived growth factor (B. BCGF), suggesting autocrine-mediated growth. The ability of IL-4 to induce proliferation of the A4 cell line was dependent on the level of autocrine growth. At low cell density, IL-4 induced marked dose-dependent proliferation. However, as A4 cell density increased, the ability of IL-4 to induce proliferation was diminished. The possibility that IL-4 may be mediating the autocrine growth of A4 cells was ruled out, because A4 cell-derived BCGF failed to induce CD23/low affinity receptors for the Fc region of IgE on activated tonsillar B cells and anti-IL-4 antibody did not block B. BCGF activity. We found that IL-4 stimulation of A4 cells and activated tonsillar B cells is associated with enhanced production of B. BCGF. These data indicate that human IL-4 has the capacity to promote proliferation of the B cell line A4, and that the ability of IL-4 to function as BCGF is associated with enhanced autocrine growth of activated B cells.
The addition of exogenous gangliosides to cultured human cells results in the cell type-specific expression of novel surface antigens by a biosynthetic process.
Authors: Furukawa K , Thampoe I J , Yamaguchi H , Lloyd K O
Published: December 2022

After the observation that human mAb 32-27M reacts only with melanoma and astrocytoma cells cultured in the presence of fetal bovine serum, a novel pathway for the uptake of exogenous gangliosides, their further biosynthesis, and expression at the cell surface as novel Ag has been elucidated. The addition of fetal bovine serum to melanoma and astrocytoma cells growing in synthetic medium (insulin-transferrin-selenium) resulted in reactivity with Ab32-27M. As antibody 32-27M detects N-glycolylneuraminic acid (NeuGc)-containing gangliosides, the effect of adding a number of different gangliosides to melanoma and astrocytoma cells cultured in the synthetic medium was studied. Only the addition of NeuGc-GM3 resulted in the development of Ab32-27M reactivity. The identity of the antigenic structures developed after addition of fetal bovine serum or NeuGc-GM3 was determined by analysis of the gangliosides from both samples. The major component detected in melanoma cell lines was shown to be N-acetylneuraminic acid-NeuGc-GD3. Another, slower moving component, present in some melanomas and in astrocytomas may be N-acetylneuraminic acid-NeuGc-GD2. The cell type specificity for these processes can be most readily explained by postulating that all cells can take up exogenous gangliosides but only melanoma and astrocytoma cells have sufficiently high levels of GM3 alpha 2----8-sialyltransferase for the conversion of added NeuGc-GM3 to disialogangliosides to be effective. These results demonstrate a novel pathway for exogenous glycolipid processing that can lead to novel Ag expression but may also play a role in normal glycolipid metabolism and function.
VH sequence of a human anti-Sm autoantibody. Evidence that autoantibodies can be unmutated copies of germline genes.
Authors: Sanz I , Dang H , Takei M , Talal N , Capra J D
Published: December 2022

The utilization of germline genes for the synthesis of autoantibodies has been suspected for many years based on the presence of cross-reactive idiotypes among patients as well as in some healthy first-degree relatives of patients with several autoimmune diseases including SLE. One such system of idiotypes involves anti-Sm antibodies, which are highly specific for SLE. To definitively establish the utilization of germline genes in the Sm system, we produced human-human B cell hybridomas from a patient with SLE who had circulating anti-Sm antibodies. One stable hybridoma designated 4B4 secretes an IgM-kappa mAb that binds Sm and shares idiotypic determinants with other anti-Sm antibodies. A second anti-Sm antibody (3C3), isolated from the same patient was also studied. Oligo(dT) priming was used to produce cDNA corresponding to full length IgM. Sequence analysis revealed that the VH gene segment (1-96) of 4B4 is identical to a VH sequence previously detected in a fetal liver cDNA library by Schroeder and his co-workers as well as a germline VH recently described by Berman and his associates. The identity of a lupus mAb and sequences derived from unrelated individuals provides strong evidence that this autoantibody is a direct copy of a germline gene.
Amino acid substitutions in VH CDR2 change the idiotype but not the antigen-binding of monoclonal antibodies to alpha(1----6)dextrans.
Authors: Sikder S K , Borden P , Gruezo F , Akolkar P N , Bhattacharya S B , Morrison S L , Kabat E A
Published: December 2022

An idiotype defined by mAb and polyclonal antibodies to 10.16.1, an anti-alpha(1----6) dextran was previously reported to be expressed on most BALB/c anti-alpha(1----6)dextrans with groove-type sites and to involved CDR3 and probably CDR2. By comparing amino acid sequences of VH and VL derived from cDNA of idiotype+ and idiotype- anti-alpha(1----6)dextran hybridoma proteins, an idiotope was assigned to VH CDR2. Substitution of phenylalanine for leucine at residue 52 in CDR2 coupled with amino acid changes at either residue 58 or residues 57 and 60 abolished expression of this idiotype without affecting Ag binding.