bluuebunny/crossref_metadata_2025 huggingface dataset:
Protein synthesis in antigen processing.
Authors: Jensen P E
Published: December 2022

Recent studies indicate that Ag pass through a chloroquine-sensitive intracellular pathway in accessory cells before they are recognized by class II-restricted T cells. Our results indicate that this is also true for insulin. Unexpectedly, we find that protein synthesis is required for optimal accessory cell-dependent processing of insulin and other proteins by adherent macrophages. Treatment of APC with inhibitors of protein synthesis, before and during exposure to Ag, inhibits their subsequent ability to activate murine T cell hybridomas. Experiments are described which suggest that this effect is localized to intracellular processing of Ag rather than uptake or presentation, per se. Inhibition is reversible, and is not observed in special situations where intracellular processing of Ag is not required. A distinct lag period is required for inhibition of processing after inhibition of macrophage protein synthesis. One possible interpretation is that protein synthesis is necessary for maintenance of a labile protein crucial for intracellular processing of Ag. Alternatively, the susceptibility of processing to inhibitors of protein synthesis may reflect an obligate intracellular association of Ag and newly synthesized class II histocompatibility molecules.
Lectin-induced phosphatidylinositol metabolism in lymphocytes is potentiated by macrophages.
Authors: Grier C E , Mastro A M
Published: December 2022

The generation and hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) have been shown to occur early after the stimulation of a variety of cells and to trigger an array of responses, including proliferation in lymphocytes. In this study we examined the requirement for macrophages in the production and metabolism of PIP2 in lectin-stimulated lymph node lymphocytes. When lymph node cells (LNC) were stimulated by the mitogenic lectin, Con A, a two- to threefold increase in [3H]inositol-labeled PIP2 occurred within 15 min, followed by a decrease to the level of unstimulated cells by 30 min. In addition, there was also a two- to threefold increase in the accumulation of [3H]inositol-labeled inositol monophosphate (IP) 30 min after the addition of lectin. However, when the LNC were depleted of macrophages by adherence to plastic and passage over cotton columns before the addition of Con A, the PIP2 response was not seen and the IP accumulation was reduced by 40%. The macrophage-depleted, Con A-stimulated cultures failed to synthesize DNA. [3H]TdR incorporation was only 15% of that of Con A-stimulated unseparated LNC cultures. Soluble IL-2 production was also depressed to 20% of that observed in macrophage-containing cultures. Furthermore, when macrophages (2%) were added to the macrophage-depleted cultures, PIP2 production, IP accumulation, and [3H]TdR incorporation were restored to levels observed in the stimulated unseparated LNC cultures. The addition of IL-1 or the phorbol ester 12-O-tetradecanoylphorbol-13-acetate in combination with Con A also restored both [3H]TdR incorporation and IL-2 synthesis in macrophage-depleted cultures. However, they did so without a detectable increase in phosphatidylinositol metabolism. Collectively, these data suggested that the production and degradation of PIP2 in Con A-stimulated lymphocytes was potentiated by the presence of macrophages.
Impairment of in vitro natural antibacterial activity in HIV-infected patients.
Authors: Tagliabue A , Nencioni L , Mantovani A , Lazzarin A , Villa L , Romano M , Rossini S , Foppa C U , Fanetti G , Masello L C
Published: December 2022

Circulating PBMC of healthy subjects possess an in vitro natural antibacterial (NA) against enteropathogenic bacteria, including Salmonella species. The effector cell of NA activity is a CD: 4+, 8-, Leu-8/TQ-1+ T lymphocyte acting against bacteria via cytophylic IgA in a mechanism similar to antibody-dependent cellular activity. Because AIDS is a profound immunodeficiency caused by HIV involving primarily CD4 lymphocytes and in particular the Leu-8/TQ-1 subset, it was of interest to assess NA activity of HIV+ subjects at various stages of the disease. Results indicate that NA activity against Salmonella typhi and Salmonella paratyphi C is significantly decreased in AIDS as well as in lymphadenopathy syndrome patients. Furthermore, sera containing IgA against salmonellae were not able to arm PBMC from HIV+ patients. The humoral response against S. typhi-LPS was also greatly decreased after HIV infection, in contrast to the known hypergammaglobulinemia seen in these subjects. Defective NA activity might contribute to the increased incidence of salmonellosis observed in AIDS.
Differential functional expression of the C8 subunits. Primary role of C8 beta in assembly of intact C8.
Authors: Densen P , McRill C M
Published: December 2022

The eighth component of C (C8) is composed of two subunits C8 beta and C8 alpha-gamma, which are non-covalently bound in a 1/1 ratio in the intact molecule. The genes encoding the polypeptide chains composing the subunits demonstrate close genetic linkage. To assess the functional expression of these genes at the protein level, normal human serum and C8-deficient sera were electrophoresed in native polyacrylamide gels following which C8, C8 beta, and C8 alpha-gamma were detected using hemolytic overlays. These experiments demonstrated that normal sera contained free C8 alpha-gamma in addition to intact C8. Free C8 alpha-gamma was not observed when C8 was reconstituted by mixing C8 beta-deficient serum with C8 alpha-gamma-deficient serum in a ratio optimized for C8 activity, suggesting that the free C8 alpha-gamma observed in normal serum was not due to dissociation of intact C8. Inasmuch as this technique did not adequately separate C8 and C8 beta, sera were also examined by anion exchange chromatography. C8 alpha-gamma-deficient serum contained C8 beta in a single peak in the 1.4 ms/cm fall through. C8 beta-deficient serum contained a major peak of C8 alpha-gamma at 7.1 ms/cm and a lesser peak coeluting with C9 at 9.5 ms/cm. Normal serum contained both intact C8 eluting between 2.4 to 5.5 ms/cm and C8 alpha-gamma eluting at 7.1 ms/cm. Free C8 beta was not detectable in normal serum indicating that free C8 alpha-gamma was not due to C8 dissociation. Mixing aliquots from the chromatographic peak of C8 beta activity with the peaks of C8 alpha-gamma activity in C8 beta-deficient serum or in normal serum generated intact C8 hemolytic activity. Non-reducing SDS-PAGE and Western blotting with anti-C8 confirmed the presence of antigenic material of appropriate m.w. in each peak. These findings demonstrate that serum contains excess C8 alpha-gamma relative to C8 beta, despite the equimolar presence of the subunits in intact C8. Thus the availability of C8 beta determines the quantity of C8 produced. Further, these data suggest the possibility that the C8 structural genes may be differentially expressed despite their close genetic linkage.
Responsiveness to human mononuclear cell-derived histamine-releasing factor. Studies of allergic status and the role of IgE.
Authors: Baeza M L , Haak-Frendscho M , Satnick S , Kaplan A P
Published: December 2022

Human mononuclear cell-derived histamine-releasing factor (MNC-HRF) has m.w. of 29,000 and 23,000 and two charge species isoelectric at pH 6.9 and 7.3. Minor forms of HRF are also seen at m.w. of 12,000 to 15,000 and 60,000 to 80,000. In this manuscript we have further defined the response of human basophils to MNC-HRF in terms of atopic status of cell donors and the role of IgE antibody. We found that basophils of allergic persons, nonallergic controls, or donors who have positive skin tests but no symptoms, cannot be distinguished based upon responsiveness to HRF. Further, the response of basophils that have been desensitized to anti-IgE is retained when they are subsequently stimulated with HRF derived from human MNC. Thus an interaction with cell-surface IgE to cause histamine release is not evident. Nevertheless, one of two IgE myeloma proteins tested were capable of binding HRF and such binding could be utilized as a purification step. We conclude that MNC-HRF is heterogeneous in terms of size, charge, and affinity for a subpopulation of IgE. Histamine release is more prominent in atopic individuals but does not appear to require an interaction with IgE.
Characterization by monoclonal antibodies of a target cell antigen complex recognized by nonspecific cytotoxic cells.
Authors: Jaso-Friedmann L , Evans D L , Grant C C , St John A , Harris D T , Koren H S
Published: December 2022

Fish nonspecific cytotoxic cells (NCC)3 recognize and lyse a large variety of human and mouse transformed cells. In an effort to determine the Ag recognized by NCC on these targets, mAb were raised against NC-37 target cells. Four anti-NC-37 mAb were chosen for further characterization based on their effects on NCC lysis of target cells. Purified mAb 18C2 and 1E7 (IgM isotype) inhibited NCC killing of the following targets: U937, MOLT-4, K562, HL-60, DAUDI, NC-37, P815, and YAC-1. The dose-dependent inhibitory activity occurred at the target cell level and ranged from 50 to 70% at a concentration of 50 micrograms/well when compared to noninhibitory mAb 7C6 and 1D4 (IgG isotype). Similarly, mAb 18C2 protected the fish parasite Tetrahymena pyriformis from lysis by NCC when compared to mAb 7C6. Adsorption experiments demonstrated that the inhibitory effect on NC-37 lysis by NCC could be removed in a titratable fashion by incubation of mAb 1E7 with any one of the other target cell lines, but it could not be removed by incubation with effector cells. The inhibitory activity of mAb 1E7 and 18C2 was shown to be caused by the inhibition of conjugate formation between effector and NC-37 target cells. The relative membrane concentration of the antigenic determinants recognized by these mAb on the target cells was studied by flow cytometry using FITC-labeled mAb. These experiments showed that all four mAb bound to the surface of the cells tested. Biochemical analysis with Western blots and immunoprecipitation showed that mAb 18C2 and 1E7 recognize two Ag in NC-37 lysates: a larger protein of around 80 kDa and a smaller one of 42 kDa.
Induction of aggregation and enhancement of proliferation and IL-2 secretion in human T cells by antibodies to CD43.
Authors: Axelsson B , Youseffi-Etemad R , Hammarström S , Perlmann P
Published: December 2022

CD43 (large sialoglycoprotein) is a heavily glycosylated protein expressed on virtually all thymus-derived lymphocytes, on a subpopulation of B cells and on granulocytes. Recently, an anti-CD43 mAb (L10) was shown to induce proliferation in T cells comparable to that induced by anti-CD3. The L10 antibody was reported to react with both sialylated and desialylated CD43. In order to further elucidate the role of CD43 in various T cell functions we have studied the biologic properties of two other mAb (B1B6 and E11B, IgG1) directed against sialic acid-dependent epitopes on CD43. Addition of low amounts of antibody (5 to 10 ng/ml) to freshly isolated T cells or to T cell lines resulted in a rapid clustering of the cells. Fab fragments were also active albeit at a 10-fold higher concentration. Aggregation was dependent on active cell metabolism (inhibited by azide and at low temperatures), on the presence of divalent cations (Mg2+) and was inhibited by antibodies to CD18 but not by antibodies to CD11a (leukocyte function-associated Ag-1 alpha). B1B6 and E11B were poorly mitogenic when added alone in soluble form to PBL or to T cells. However, supernatants from cultures of PBL treated with B1B6 for 2 days contained IL-2 activity. No increase in the number of CD25+ cells was seen during the same period. Exogenously added IL-2 did not synergize with B1B6 or E11B in activation of PBL, whereas proliferation was significantly increased by the addition of the antibodies to activation systems with low endogenous production of IL-2 (PMA or soluble anti-CD3). The anti-CD43 antibodies amplified T cell proliferative responses induced by Con A or leukoagglutinin from Phaseolus vulgaris. F(ab')2 fragments enhanced proliferation significantly better than Fab fragments suggesting that cross-linking of CD43 molecules was an essential features of the amplifying signal. Compared with cultures activated by Con A alone, an increased number of CD25+ cells and of blast cells as well as an increased IL-2 production was observed in cultures activated by B1B6-Con A. The results indicate that regulatory signals, which may function to modify homo- or heterotypic T cell adhesion as well as autocrine production of IL-2, can be transduced through CD43.
Degradation of a monoclonal anti-mu chain antibody in a human surface IgM-positive B cell line starts in prelysosomal vesicle.
Authors: Ruud E , Kindberg G M , Blomhoff H K , Godal T , Berg T
Published: December 2022

The surface IgM-mediated endocytosis and intracellular transport of an anti-F(c mu)5 mAb was studied by using subcellular fractionation in sucrose gradients. The results of such experiments showed that antibody was initially endocytosed in vesicles of low density, and later transferred to a presumably lysosomal compartment of higher density. SDS-PAGE analysis of gradient fractions showed that high Mr degradation fragments of the endocytosed antibody were formed in the low density vesicles before terminal degradation could be recorded. The partial degradation of the antibody was not blocked by low temperature or enzyme inhibitors, such as leupeptin and benzyloxycarbonyl-phenylalanylalanine-diazomyethyl-ketone, all of which severely retarded terminal degradation. The data also suggested that the recycling of partially degraded antibody to the cell surface employed a pool of such low density prelysosomal vesicles.
IL-1 signaling for IL-2 production in T cells involves a rise in phosphatidylserine synthesis.
Authors: Didier M , Aussel C , Pelassy C , Fehlmann M
Published: December 2022

Activation of Jurkat T cells with anti-TCR, anti-CD3, anti-CD2, or PHA is accompanied by a strong inhibition of phosphatidylserine (PS) synthesis. The inhibition of the synthesis of this phospholipid could be partially reversed by IL-1. In Jurkat cells, IL-1 did not activate phosphodiesterases as demonstrated by the lack of change of inositol triphosphate and diacylglycerol levels as well as the lack of change in cytosolic Ca2+ concentration. Furthermore, IL-1 did not modify the intracellular level of cGMP and cAMP, suggesting that the observed rise of PS synthesis could play the role of mediator IL-1 action. As PS is a necessary cofactor for the activation of protein kinase C, our results suggest strongly that IL-1 modulate protein kinase C activity in the activated lymphocyte through its action on PS synthesis.
Endocytic pathway of recombinant murine tumor necrosis factor in L-929 cells.
Authors: Mosselmans R , Hepburn A , Dumont J E , Fiers W , Galand P
Published: December 2022

The fate of TNF after binding to the surface of L-929 cells was followed by using murine rTNF coupled to colloidal gold as a probe. A time-course study using electron microscopy was performed. Our results confirm previous indications obtained from biochemical studies suggesting that TNF is internalized by this cell type. They further directly show that internalization proceeds through the classical receptor-mediated endocytosis pathway, i.e., via clathrin-coated structures and endosomes before accumulation in secondary lysosomes.
Relative inefficiency of terminal complement activation.
Authors: Bhakdi S , Fassbender W , Hugo F , Carreno M P , Berstecher C , Malasit P , Kazatchkine M D
Published: December 2022

The efficiency of generation of fluid-phase SC5b-9 and membrane C5b-9(m) complexes relative to cleavage of C3 and C5 was studied. Fluid-phase C activation was induced through addition of purified bacterial Ag to human serum. Sephadex beads were used as particulate activators of the alternative pathway. Rabbit or antibody-coated sheep or human E were used to study formation of cytolytic C5b-9(m) complexes. The molar ratios of C3a:C5a generated in the model systems were found to be in the range of 60 to 200:1 in the case of soluble immune complex activators, and 70 to 150:1 with particulate activators and cells. The efficiency of C5 cleavage relative to C3 cleavage increased on surfaces with the density of antibody and/or C3b-binding sites. With soluble immune complexes, the efficiency of subsequent SC5b-9 generation displayed wide variations dependent on Ag and donor with molar ratios of C5a:SC5b-9 ranging from 30:1 for teichoic acid and sometimes approaching 1:1 for streptolysin-O. In contrast, activation on particles or cells always led to C5a:C5b-9 (calculated as the sum of generated moles SC5b-9 and C5b-9(m] ratios approaching 1:1. Hence, there is an overall inefficiency of terminal sequence activation in the C cascade due first to a dissociation at the level of C5 convertase formation/C5-cleavage and second, to a frequent inefficiency of C5b-utilization in the fluid-phase. The results provide an explanation for the very low levels of SC5b-9 found in plasma of healthy individuals and in patients with C-consuming immune complex disease.
Coxsackievirus B-3-induced myocarditis. Virus and actinomycin D treatment of myocytes induces novel antigens recognized by cytolytic T lymphocytes.
Authors: Huber S A , Heintz N , Tracy R
Published: December 2022

BALB/c mice inoculated with 6 x 10(4) plaque-forming units of a myocarditic variant of Coxsackievirus, group B, type 3 (CVB3M) developed three distinct CTL populations recognizing putative virion (virus specific CTL, VCTL), normally expressed heart (autoreactive CTL, ACTL), and aberrant metabolic (MCTL) Ag. Induction of the MCTL-specific Ag on cardiocytes correlated with the ability of the myocarditic viruses and actinomycin D to interfere with cellular metabolism as measured by 3H-uridine and 3H-leucine incorporation. ACTL specifically lysed uninfected cardiocyte targets, but ACTL Ag expression was lost soon after infection of monocytes only to reappear 6 h later. MCTL and VCTL could be separated by adsorption to myocytes treated with either actinomycin D- or UV-inactivated CVB3M. MCTL cross-reactively lysed myocytes infected with another virus suppressing 3H-uridine incorporation (encephalomyocarditis virus) but failed to react to targets infected with viruses not inhibiting cell metabolism. VCTL specifically lysed only cells infected with the homologous virus. ACTL belonged to the CD8 (Lyt-2+) T cell subset, whereas both VCTL and MCTL were CD4 (L3T4+) T cells.
T cell-induced Ig allotypic suppression in mice. II. Both CD4+ CD8- and CD4- CD8+ T cell subsets from sensitized Igha mice are required to induce suppression of Igh-1b allotype expression.
Authors: Benaroch P , Bordenave G
Published: December 2022

We established the phenotype of T splenocytes (Ts) from Igha/a BALB/c mice sensitized against B splenocytes from the Ighb/b CB20 congenic mice that induce Igh-1b (IgG2a of the Ighb haplotype) suppression. This was achieved by studying the action of anti-T cell subset mAb on the capacity of Ts to induce this chronic allotypic suppression in Igha/b (BALB/c x CB20)F1 mice. The Ts were treated with cytotoxic anti-mouse CD4 or anti-mouse CD8 rat mAb in vitro before their injection into the Igha/b newborns or in vivo after their injection into the Igha/b newborns. Exposure to either anti-CD8 or anti-CD4 mAb in vitro or in vivo leads to loss of the capacity of Ts to induce Igh-1b allotypic suppression. Mixing CD4+-cell-depleted Ts and CD8+-cell-depleted Ts preparations restored the capacity of the cells to induce Igh-1b suppression. Thus, both CD4+ CD8- Ts and CD4- CD8+ Ts are required for the induction of this allotypic suppression. Bone marrow cells and B splenocytes from Igh-1b-suppressed adult Igha/b mice were shown to be able to durably express Igh-1b when transferred into irradiated Igha/a BALB/c hosts whereas whole spleen cells from such donors failed to do it. Abrogation of Igh-1b suppression by in vivo anti-CD8 mAb treatment was achieved in adult Igha/b heterozygotes but with a lower efficiency than in adult Ighb/b homozygotes, all being chronically Igh-1b suppressed. The CD4- CD8+ cell population essential for maintaining this suppression is resistant to in vivo 600 rad irradiation and seems to be slightly inhibited by in vivo administration of free Igh-1b.
Regulation of IL-1 and TNF-alpha expression during the differentiation of bone marrow derived macrophage.
Authors: Myers M J , Pullen J K , Ghildyal N , Eustis-Turf E , Schook L B
Published: December 2022

Macrophage differentiation is accompanied by the acquisition of both Ag presentation and tumoricidal activities. In this set of experiments, the expression of IL-1 by bone marrow-derived macrophage (BMDM) was found to be highly regulated, with both the expression of IL-1 mRNA and mIL-1 appearing only at discrete stages of activation. The accumulation of IL-1 alpha (membrane) mRNA was induced by endotoxin but not IFN-gamma or CSF-1. mIL-1 was detected by D10.G4.1 T cells on BMDM only after 7 days of in vitro differentiation. Secreted IL-1 beta was detected by day 3 of culture, with enhanced production observed after activation with endotoxin. IL-1 beta mRNA was found to be constitutively expressed in BMDM as early as day 3 of culture. The expression of IL-1 beta mRNA was up-regulated by endotoxin after 30 min of exposure with maximal expression occurring after 2 to 6 h of exposure. Constitutive expression of IL-1 alpha mRNA was not detected but 1 h of endotoxin exposure resulted in the appearance of IL-1 alpha transcripts. As with IL-1 beta, TNF-alpha mRNA was also constitutively expressed during a wide time period of differentiation; however, in contrast, to IL-1 beta, TNF-alpha mRNA expression was up-regulated by both endotoxin and IFN-gamma. The expression of TNF-alpha macrophage by BMDM coincided with the acquisition of tumoricidal activity. An examination of the mRNA sequences encoding the proto-oncogenes c-myc and c-fms demonstrated the expression of c-myc only on day 3, whereas c-fms was constitutively expressed throughout the culture period. Endotoxin stimulation of BMDM resulted in a transitory increase in c-myc expression only at day 3 of culture, whereas endotoxin had no effect on c-fms expression until 7 days of culture at which time expression declined. In contrast, the expression of transferrin receptor mRNA transcripts, which were also constitutively expressed throughout the entire culture period, were not affected by stimulation with either endotoxin or IFN-gamma. These results indicate BMDM expression of the affector molecules IL-1 alpha and beta and the effector molecule TNF-alpha are regulated separately during unique "differentiation-specific" phases of development.
Role for the third constant domain of the IgG H chain in activation of complement in the presence of C1 inhibitor.
Authors: Okada M , Utsumi S
Published: December 2022

The multidomain architecture of Ig H chains was initially implicated in the variety of functions imposed on each species of Ig. However, the activation of C by IgG is the only function that has been attributed to a single domain of C gamma 2, whereas most of other functions of IgG require both C gamma 2 and C gamma 3 domains. This one domain-one function relationship in the C activation by IgG, too, was questioned recently by the fact that a C gamma 3-less fragment of rabbit IgG, F(acb)2, is definitely less capable of activating C than intact IgG. Here we reexamined capacities of F(acb)2 to bind and activate C1 in the presence and absence of C1 inhibitor (C1-In) in comparison with intact IgG, by using SRBC sensitized with these proteins (EFacb, EIgG). At an ionic strength of 0.065 and 37 degrees C, where C1q was bound equally well by these cells and the dissociation was limited, C1s, presumably in the form of C1r2C1s2, dissociated from EFacb at a rate 7-fold greater than that from EIgG, irrespective of the presence or absence of C1-In. A physiologic concentration of C1-In reduced the rate of C1 activation by EFacb to 5% that by EIgG. The results present evidence that the C gamma 3 domain, too, plays a crucial part in the C1 activation process by stabilizing the zymogenic conformation of C1 and protecting it from the attack by C1 inhibitor.
Purification and analysis of an antigen-specific suppressor factor from a T cell hybridoma specific for phenyltrimethylamino hapten.
Authors: Webb D R , Semenuk G , Krupen K , Jendrisak G S , Bellone C J
Published: December 2022

The synthetic monovalent antigen L-tyrosine-p-azophenyltri-methylammonium (tyr (TMA)) induces in A/J mice, a cascade of regulatory T cells in the absence of any detectable effector function (e.g., CTL, delayed-type hypersensitivity, etc.). An important component of the activated T cells is a first order suppressor T cell or Ts1 that is Ly-1+2-, functions only at the afferent limb of the anti-TMA response, binds the TMA ligand and bears cross-reactive idiotypes associated with anti-TMA antibodies. This Ts1 produces a suppressor factor (TsF1) that binds the TMA ligand, bears the cross-reactive idiotypes and I-J determinants and functions to induce an idiotype-specific Ts2 population. To study the biochemistry of this TsF, use was made of T cell hybridomas that constitutively produce TMA-TsF1 (8A.1 and 8A.3). The TsF1 was purified from culture supernatant or cell extracts by (NH4)2SO4 precipitation, reverse phase HPLC and either affinity chromatography or by preparative IEF. The TsF1 has an isoelectric point of 6.5 and a m.w. of 26,000 or 62,000 as analyzed by SDS-PAGE or high performance molecular sieve chromatography. Its precipitation in 30 to 40% (NH4)2SO4; elution pattern from reverse phase high performance columns; its capacity to bind to a mAb specific for L-glutamic acid 60L-alanine30-L-tyrosine10 (GAT)-TsF1 strongly suggest that this protein belongs to the same family of proteins as do the GAT-TsF1 described previously. Most noteworthy is that although these TsF1 proteins show remarkable similarities, they are absolutely specific in their biologic activity; TMA-TsF1 will not suppress the response to GAT-BA-TNP and GAT-TsF1 will not suppress the response to TMA-BA-TNP. Thus the TMA-TsF1 represents a second example of a unique group of Ag-specific proteins whose function is to induce or activate other suppressor T cells in the primary immune response to Ag.
Alterations in the antigenic structure of two major HSV-1 glycoproteins, gC and gB, influence immune regulation and susceptibility to murine herpes keratitis.
Authors: Hendricks R L , Tao M S , Glorioso J C
Published: December 2022

We previously demonstrated that anterior chamber (AC) injection of HSV-1 before or simultaneous with topical corneal HSV-1 infection resulted in cellular immune tolerance of HSV-1 Ag and a reduced frequency of corneal stromal lesions. In the present study, we have investigated the role of the HSV-1 cell-surface glycoproteins gC and gB in the induction of tolerance, and the resulting reduced susceptibility to HSV-1 corneal stromal disease. These studies utilized mutant strains of HSV-1 with deletion or point mutations in the gene coding for gC or gB. Groups of mice received topical corneal infections with wild-type HSV-1, followed by AC injection of the same eye with wild-type HSV-1 or a mutant strain. Varying the antigenic composition of the virus injected into the AC resulted in three distinct patterns of immune responsiveness. In agreement with our previous findings, AC injection of wild-type HSV-1 induced a state of HSV-1 specific tolerance that extended to both the delayed type hypersensitivity (DTH) and CTL responses. A mutant strain lacking gC (gC-) induced partial tolerance characterized by undetectable CTL activity but a normal DTH response. A mutant strain lacking gB (gB-) caused partial suppression of the CTL response and no reduction of the DTH response. Thus, whereas gB may be involved in CTL tolerance induction in this model, gC clearly is not involved. In contrast, both gC and gB must be present in the AC to induce detectable DTH tolerance. The latter interpretation was strengthened by the observation that AC injection of a mixture of gC- (expressing normal gB) and gB- (expressing normal gC) effectively suppressed the DTH response to wild-type HSV-1. A panel of mar mutants with individual point mutations affecting gC and gB was used to identify the epitopes responsible for induction of DTH tolerance. Two of the gC mutants failed to induce DTH tolerance to wild-type HSV-1 when injected into the AC, suggesting that the sites on the gC molecule that are altered by these mutations are important for the induction of DTH tolerance. Similarly, one of the mar mutants for gB uniformly failed to suppress the DTH response, while another had a variable effect. The unique pattern of cellular immune reactivity exhibited by the mice receiving simultaneous topical corneal infection with wild-type HSV-1 and AC injection of gC- (no CTL but normal DTH) was associated with significantly reduced susceptibility to HSV-1 corneal stromal lesions.(ABSTRACT TRUNCATED AT 400 WORDS)
Idiotype variants emerging after anti-idiotype monoclonal antibody therapy of a murine B cell lymphoma.
Authors: Weiner G J , Kaminski M S
Published: December 2022

One of the difficulties encountered with the treatment of human B cell malignancies with anti-Id antibodies is the emergence of Id variants. The current study was designed to investigate this phenomenon further by using the murine B cell lymphoma model 38C13. Tumors were harvested that developed despite treatment with the anti-Id antibody S1C5 in mice inoculated with 38C13 cells and evaluated by immunofluorescence. Various phenotypes were found among escaping tumor cells. Some cells continued to react with S1C5 whereas others lost S1C5 reactivity. Among these latter cells, some continued to express surface IgM kappa, whereas others no longer expressed surface mu or kappa. After Id variant cell lines were established, immunofluorescence and ELISA of cell lysates from the surface IgM kappa- lines revealed persistent intracellular mu H chain but no detectable kappa. Surface IgM kappa+ lines were fused with myeloma cells and the Ig proteins secreted by the resultant hybridomas analyzed. The apparent m.w. of the mu-chains of these rescued Ig was the same as wild-type 38C13, whereas the kappa-chains were either the same or different in m.w. from the wild type. The IgM kappa of the variant line, T3C, weakly reacted with S1C5 and did not react with other anti-Id antibodies. The IgM kappa of the other variants were nonreactive with all the antibodies. Immunofluorescence of these surface Ig+ variants confirmed this finding. Some of the surface Ig+ and Ig- variant lines grew identically to wild-type tumor in vivo, but only the weakly S1C5-reactive variant T3C was inhibited in its growth by S1C5. Moreover, T3C was the only one of these lines capable of being lysed in vitro with S1C5 by antibody-dependent cellular cytotoxicity. Further studies revealed that surface Ig+ and Ig- variants emerge in escaping tumors with similar frequency and that these variants represent a major mode of tumor escape from anti-Id treatment in this model.
Selective loss of HLA-A or HLA-B antigen expression in colon carcinoma.
Authors: Momburg F , Ziegler A , Harpprecht J , Möller P , Moldenhauer G , Hämmerling G J
Published: December 2022

A panel of colorectal carcinomas has been examined immunohistologically with mAb specific for allodeterminants of HLA-A or HLA-B, and with mAb reactive with HLA-A,B,C framework determinants or beta 2-microglobulin. Out of 85 carcinomas, 5 cases were completely negative for all class I Ag in the tumor cell population. Fifteen cases exhibited a differential expression of HLA-A and HLA-B products in all tumor cells or in a subset. Such tumor cells showed strong staining with monomorphic HLA antibodies but failed to stain with certain allospecific mAb. Negativity of tumor cells was scored only when normal stromal cells within the tumor mass were clearly labeled, indicating expression of the particular allodeterminant(s) in the given tissue. Four carcinomas showed a selective loss of HLA-A2 or a non-A2 HLA-A allospecificity in all tumor cells or a major subset, whereas HLA-B Ag were expressed. Seven cases showed a selective loss of the HLA-Bw6 superspecificity in the tumor cell population; HLA-Bw4 and HLA-A Ag were present. Three cases exhibited a combined loss of HLA-A2 and HLA-Bw6 Ag, with non-A2 HLA-A and HLA-Bw4 Ag being expressed. A further case was HLA-Bw6/Bw4 negative in a major tumor cell subset and HLA-A negative in the complementary minor subset. The results show that a considerable proportion of colorectal carcinomas is heterogeneous with regard to HLA-A and HLA-B expression. The reasons for the appearance of tumor subsets with complete loss of class I Ag or selective loss of only HLA-A or HLA-B Ag are not clear, but it is conceivable that some of them arose because they have escaped immunoselection.
The expression of T cell receptor-associated proteins during T cell ontogeny in man.
Authors: Campana D , Janossy G , Coustan-Smith E , Amlot P L , Tian W T , Ip S , Wong L
Published: December 2022

The expression of TCR-associated molecules was examined in human fetal and postnatal tissues. From gestational wk 7 onward in the fetal liver, putative prothymocytes have been identified with cytoplasmic CD3 positivity (cCD3+). These immature cells are TdT- and do not express membrane CD3 (mCD3-) or TCR beta identified by beta F1, but show CD7 and CD45 positivity without CD1, CD2, CD5, CD4, CD8, CD10, and class II Ag. Their high proliferative activity is indicated by greater than 85% Ki67 positivity. After the 10th wk, beta F1+, mCD3+ cells also appear in the liver and these are mostly Ki67- but no TCR gamma delta-bearing cells can be identified at such an early stage of extrathymic development. In the mCD3- TdT-fetal thymus (10 1/2 to 18th wk) cCD3+, mCD3- CD1-blasts proliferate (Ki67+) and lack TCR-beta or TCR-gamma delta. The TdT-, CD1+ cortical thymocytes develop into TCR-beta + and WT31-positive (TCR-alpha beta +) cells. Subsequently TdT-positive thymocytes become detectable around 19 to 20 wk, and in such glands the peak of proliferative activity is seen among TdT+, cCD3+ cells which appear to acquire, in a regular sequence, cytoplasmic beta F1 (TCR-beta), mCD3, and TCR-alpha beta (WT31 positivity) together with the loss of TdT and Ki67 positivity. A newly described transitional population of cells is TdT-, beta F1+ but exhibits no detectable WT31 positivity. These cells correspond to the CD1+, mCD3+ thymocytes and are probably the targets of thymic selection. The cells of the TCR-gamma delta lineage, detected by mAb TCR-delta-1 and delta TCS1, are rare (0.02 to 0.5%) among thymocytes from gestational wk 10 1/2 onward through the whole span of thymic development, but these cells include a proportion (18 to 59%) of cells expressing CD1 Ag, suggesting that these TCR-gamma delta cells differentiate in the thymus. Among the CD1+, TCR-gamma delta + thymocytes, no TdT positivity can be detected.
Immortalization of EBV-infected B cells is not influenced by exogenous signals acting on B cell proliferation. Effects of mutant EL-4 thymoma cells and transforming growth factor-beta.
Authors: Straub C , Zubler R H
Published: December 2022

We have recently developed a culture system in which 90% of B cells from human peripheral blood or spleen are induced to strongly proliferate and generate short-term clones of a mean of about 400 antibody-secreting cells. B cells are stimulated by mutant EL-4 thymoma cells in conjunction with T cell supernatant. In the present study, we first investigated whether the frequency of B cell immortalization by EBV would be higher in this system than in a conventional system by using PBMC as fillers. The results showed that the EBV-dependent cloning frequency (0.7%) was not increased compared with the system with the use of PBMC (2.1%). However, the short term proliferation of EBV-infected B cells was 20 times increased in the EL-4 system and EBV nuclear Ag-positive cells participated in this response. Recent reports showed that transforming growth factor-beta (TGF-beta) inhibited the growth of normal B cells, whereas the growth of EBV-immortalized (lymphoblastoid) cells was not inhibited. We have studied the effects of this cytokine in the EL-4 system. TGF-beta (200 pg/ml) was found to inhibit the proliferative response of normal B cells by greater than 95% and the short term response of EBV-infected B cells by 78%. In contrast, the EBV-dependent long term cloning frequency, as well as the proportion of clones producing IgM, IgG, or IgA, was not influenced at all by TGF-beta. In conclusion, potent modulation of the early proliferative response of an EBV-infected B cell population by either stimulatory or inhibitory exogenous signals did not influence the number of cells that subsequently became immortalized.
Induction of class II MHC antigen expression in macrophages by Mycoplasma species.
Authors: Stuart P M , Cassell G H , Woodward J G
Published: December 2022

Several different Mycoplasma species have been shown to act as mitogens for either T or B cells and as stimulators of macrophage tumoricidal activity. In this report, we show that at least five different species of Mycoplasma are capable of inducing class II MHC expression on macrophages. We have observed significant induction of class II MHC surface expression on the myelomonocytic cell line, WEHI-3, as early as 24 h after deliberate infection of cultures, reaching maximal levels by 4 days. This induction was also apparent at the mRNA level as assessed by Northern blot analysis by using A alpha, E alpha, and A beta probes. However, unlike many other previously described MHC-inducing agents, mycoplasmas failed to induce class I MHC expression at either the cell surface or mRNA levels. Kinetic analysis revealed that induction of class II mRNA by mycoplasmas was slower than induction by IFN-gamma requiring 24 h rather than 8 h for significant increases to be noted. Induction by mycoplasmas does not require the presence of live organisms and remains active after heat treatment of 90 degrees C for 30 min. We have also demonstrated that mycoplasma infection of primary bone marrow macrophage cultures leads to the induction of both class I and class II genes and, as in the case of WEHI-3, this induction does not require the presence of live organisms. These data indicate that several Mycoplasma species have the capacity to induce class II MHC expression in WEHI-3 and both class I and class II MHC expression in bone marrow macrophage cultures in the absence of any T cell products.
IL-2 secretion is pertussis toxin sensitive in a T lymphocyte hybridoma.
Authors: Stanley J B , Gorczynski R M , Delovitch T L , Mills G B
Published: December 2022

Interaction of specific ligands with TCR initiates a cascade of biochemical events which leads to expression of high affinity IL-2R and subsequent IL-2 secretion. Activation of phospholipase C (PL-C) is considered to be a key event in the initiation of this cascade. However, in addition to this PL-C-dependent pathway, PL-C-independent pathways have been hypothesized. Identification of the steps constituting these PL-C-independent pathways has been difficult because activation of PL-C and the subsequent cascade of events mask the effects of such pathways. Specific inhibitors for PL-C, or mutants defective in, the PL-C pathway would facilitate delineation of alternative activation pathways. We have identified a murine pork insulin/IAd-specific T cell hybridoma, B8P3.11, in which perturbation of the B8P3.11 TCR by either Ag in association with Ia, anti-CD3 antibodies, or a mitogenic lectin does not induce increases in myo-inositol 1,4,5-triphosphate production or cytosolic free calcium, yet it does lead to IL-2 secretion. Treatment of B8P3.11 with pertussis toxin, at concentrations which ADP-ribosylate GTP-binding proteins, inhibits IL-2 secretion. Thus, signal transduction resulting in IL-2 secretion by B8P3.11 likely involves a G protein. In contrast, TCR/ligand interaction activates the PL-C-dependent pathway in LBRM 331A5, a T cell lymphoma. Furthermore, pertussis toxin treatment, which blocks IL-2 secretion by B8P3.11, does not alter IL-2 secretion by LBRM 331A5. However, similar pertussis toxin substrates are present in both cells. Therefore, B8P3.11 T cells should help to elucidate PL-C-independent activation pathways.
A single VH gene is utilized predominantly in anti-BrMRBC hybridomas derived from purified Ly-1 B cells. Definition of the VH11 family.
Authors: Hardy R R , Carmack C E , Shinton S A , Riblet R J , Hayakawa K
Published: December 2022

By establishing hybridomas from two distinct surface IgM+ splenic B cell populations, Ly-1 B cells and "conventional" (Ly-1-) B cells, we found that the Ly-1 B population includes a 30 to 70 times higher frequency (1 to 2%) of cells with specificity for bromelain treated autologous red blood cells (anti-BrMRBC) when compared with conventional B cells (0.03%). We cloned and sequenced the V genes encoding anti-BrMRBC antibody from two hybridomas made with Ly-1 B cells sorted from the spleen of SM/J mice. The VH sequence (for both) is identical with the previously reported sequence associated with this specificity and belongs to a new VH gene family. This gene family, defined here as VH11, has only two members and is the predominant VH rearranged in a collection of Ly-1 B derived anti-BrMRBC hybridomas, always in association with a single VL gene (a member of the V kappa 9 family). Furthermore, analysis of hybridomas made with Ly-1 B cells sorted from the peritoneum reveals a yet higher increased frequency of VH11-encoded anti-BrMRBC specificity (30%). This variation in frequency of anti-BrMRBC in the Ly-1 population depending on location, together with the repeated association of VH11 with a particular V kappa gene suggest that antigen driven selection is (at least in part) responsible for the biased V gene expression seen in this population. Furthermore, a mechanism that might contribute to biased expression, preferential rearrangement due to close proximity to J (as seen in pre-B lines), is excluded by localization of VH11 5' to several of the more J-proximal families (Q52, 7183).
Isolation and characterization of a cDNA encoding the KS1/4 epithelial carcinoma marker.
Authors: Perez M S , Walker L E
Published: December 2022

The mAb KS1/4 recognizes a novel cell surface glycoprotein on a variety of epithelial carcinomas which may be a useful target Ag for antibody-directed diagnostic and therapeutic approaches. Here we report the isolation and characterization of a full length cDNA clone coding for the KS1/4 Ag, as well as, physical and biochemical studies on the antigen derived from an adenocarcinoma of the lung cell line. Affinity purification of the KS1/4 Ag reveals three glycosylated species by NaDodSO4 PAGE with molecular weights of 42, 40, and 35 kDa. The 42- and 40-kDa species are similar at the protein level, differing by their degree of glycosylation, and the 35-kDa protein results from a dibasic proteolytic cleavage of the larger m.w. species. Although both the 42- and 40-kDa forms are found on the cell surface, the 40-kDa protein appears to be the predominant species. A cDNA clone containing the complete KS1/4 coding sequence and the 5'- and 3'-non-translated regions was isolated from a library constructed from the human adenocarcinoma of the lung derived cell line, UCLA-P3. The cDNA clone contains an open reading frame of 314 amino acids which includes a putative signal sequence of 23 amino acids. Northern blot analysis shows a single RNA species of 1.5-kb. Sequence analysis of the 5' and 3' noncoding regions of the KS1/4 cDNA revealed homologies to known proto-oncogenes and inflammatory mediators.
Generation and characterization of continuous lines of CD8+ suppressor T lymphocytes.
Authors: Aune T M , Pogue S L
Published: December 2022

The ability to grow normal T lymphocytes in long term culture has advanced our understanding of T cell biology. The growth of CD4+ cell lines allowed a further evaluation and appreciation of functional subtypes within this group. Cytotoxic CD8+ T cells have been characterized as well. The routine and continuous culture of Ag-nonspecific CD8+ Ts cells has been difficult to achieve. We have found that CD8+ T cells that suppress T cell proliferation and lack cytotoxic activity against T cells can be routinely obtained from PWM or PHA-stimulated PBMC. Continuous culture of T cell blasts from PWM or PHA-stimulated PBMC resulted in the growth of CD4+ and CD8+ T cells. These lines developed suppressor cell activity within 7 days after stimulation with PWM and 3 to 4 wk after stimulation with PHA. Concomitant with the development of suppressor activity was the loss of CD4+ T cells resulting in homogeneous lines of CD8+ suppressor cells. These cell lines have been maintained in continuous culture for greater than 6 mo by addition of rIL-2 twice weekly and restimulation with feeder cells and PHA every 2 wk. Activity of these cell lines was relatively resistant to irradiation or treatment with mitomycin C. Both cell lines suppressed proliferation of autologous or heterologous CD4+ T cells stimulated with PWM, OKT3, or tetanus toxoid but failed to suppress proliferation of CD4+ T cells in a mixed lymphocyte reaction. CD4+ T cells stimulated with PWM produced equivalent amounts of IL-2 in the presence or absence of Ts cells but failed to express the IL-2R (TAC) on their surface in the presence of Ts cells. By contrast, CD4+ T cell lines or cytotoxic CD8+ T cell lines failed to suppress proliferation of CD4+ T cells. With these results we describe methods for the generation and continuous culture of Ag-nonspecific CD8+ Ts cells and define some of their properties. These cells lines should be helpful in further elucidating the functional and phenotypic repertoire of CD8+ Ts cells.
Immune dysfunction associated with graft-vs-host reaction in mice transplanted across minor histocompatibility barriers. II. Reversible defect in T-dependent antibody responses.
Authors: Budhecha S , Hamilton B L
Published: December 2022

B cell and Th cell functions were assessed in mice undergoing a graft-vs-host reaction (GvHR) in response to minor histocompatibility Ag by using the plaque-forming cell (PFC) response to the T-independent Ag TNP-Brucella abortus and the T-dependent Ag TNP-SRBC. Bone marrow plus spleen cells from B10.D2 mice were transplanted into lethally irradiated B10.D2 (syngeneic recipient) or H-2d-compatible BALB/c (allogeneic recipient) to produce a chronic form of GvHR. BALB/c recipients of an allogeneic transplant demonstrated a marked and proportional lymphoid depletion of the spleen with normal percentages of B cells, T cells, and CD4+ and CD8+ T cell subsets. Mice with GvHR made normal numbers of PFC/10(5) spleen cells in response to the T-independent Ag, but a significantly depressed number of PFC/10(5) spleen cells to the T-dependent Ag compared with normal B10.D2 mice and with irradiated B10.D2 recipients of syngeneic B10.D2 marrow plus spleen cells. Mice undergoing the minor Ag GvHR made significantly larger numbers of PFC/10(5) spleen cells after secondary immunization with TNP-SRBC compared with controls. In vitro assays demonstrated that B cells from mice with GvHR responded to T help from normal B10.D2 mice and that T cells from mice with GvHR provided help to normal B cells after in vivo immunization. These data demonstrate that radiation chimeras with GvHR in response to minor histocompatibility Ag have relatively normal B cell function and an apparent defect in T helper cell function that is reversible by immunization with appropriate Ag.