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New short-lived strains of mice (SAM-P), which have been developed by Takeda et al. (1981), show a defective antibody response to T dependent (TD) antigen in vitro, as demonstrated in the accompanying paper (see page 419). In the present study, we investigated the cellular site of the defect, using a cell culture system. In this paper, it is demonstrated that T-helper (Th) cell activity for the antibody response to TD antigen is impaired, while other cellular immune responses, e.g. mixed leucocyte reaction, cytotoxic T-lymphocyte response, and delayed-type hypersensitivity reaction, are normal. These results suggest that the defect in T-helper subset is limited in helper function for the antibody response, and that the helper function for the cell-mediated immune responses is intact. These two functions of the T-helper subset are apparently regulated in a different manner. The SAM-P strains of mice may thus serve as an appropriate model for studying functional heterogeneity in T-helper/inducer cell subsets. [ABSTRACT FROM AUTHOR]

The first paper in this series reported that it was possible to reconstitute the response of low concentrations of mouse spleen lymphocytes to concanavalin A (Con A) by adding small numbers of peritoneal exudate cells (PEC) to the cultures. In this paper it is shown that the role of the PEC in this system can be partially replaced by conditioned medium (CM) prepared from PEC cultures or completely replaced by CM taken from lymphocytes cultured at optimal concentration. These CM were inactive unless fresh Con A was added to the assay cultures. Activity was present in CM which was incubated with lymphocytes or PEC for the shortest possible time but maximal activity was found after 24 hr of incubation. Activity was also found in CM prepared in the absence of Con A. Only in the case of lymphocytes cultured at optimal concentration for 24 hr was there substantially more activity in the CM thus prepared if Con A was present. PEC preparations depleted of T lymphocytes produced as much activity in CM as the untreated control. CM produced by PEC was less sensitive to heat treatment or to freezing and thawing than that produced by lymphocyte cultures. [ABSTRACT FROM AUTHOR]

Several hybridoma cell lines secreting NP-specific, murine IgE antibodies were generated by fusion of P3-X20 (γ,κ) tumour cells with spleen cells from (BALB/c x C57Bl/6)F1 (CB6F1) mice previously immunized with NP-ovalbumin. Four subclones (designated NP-ε-3.57, NP-ε-15,88, NP-ε-91,58 and NP-ε-95,31) were propagated in vivo and milligram quantities of the corresponding IgE antibodies were purified from ascitic fluid by gel filtration, ion exchange chromatography and affinity chromatography. Immunological analyses and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDSPAGE) indicated that NP-ε-15,88, NP-ε-91,58 and NP-ε-95.31 all possessed λ1 (or possibly λ3) light chains; and that NP-ε-3,57 possessed λ2 light chains; NP-&elipson;-95,31 also expressed the P3-X20 derived, MOPC-21 κ light chain. Radioallergosorbent test (RAST) titration curves, generated from the interaction of the four monoclonal IgE antibodies with NP-BSA attached to paper discs (NP-BSA-P) were found to be non-overlapping. Measurements of the relative amounts of NP-ε-aminocaproic acid (NP-CAP) and 4-hydro-3-iodo-5-nitrophenylacetyl-ε- aminocaproic acid (NIP-CAP) that were required to inhibit by 50% the binding of the 4 IgE antibodies to NP-BSA-P indicated that these antibodies were all heteroclitic, since their affinity for NIP appeared to be higher than their affinity for NP. These results, in conjunction with other findings reported in the literature, suggested that the V regions of NP-specific IgE antibodies are similar to the V regions of NP-specific IgM and IgG antibodies, produced by the same mouse strains. Finally, in vitro histamine release measurements demonstrated that two of these monoclonal IgE antibodies could mediate antigen induced histamine release from passively sensitized rat peritoneal mast cells. [ABSTRACT FROM AUTHOR]

We previously reported that incubation of human peripheral blood mononuclear cells (PBMC) for 5 days with T-cell growth factor (ICGF) resulted in lymphokine-activated killer activity against endothelial cells (EC). In this paper we report on the effects of short-term incubation of PBMC with lymphokines. We show that incubation of PBMC with lymphokines during an 18-hr period is sufficient to generate a strong cytolytic response against EC. The cytolytic capacity of the effector cells was directly dependent on the dose of lymphokine added during the induction phase. When PBMC were separated into adherent and non-adherent cells, the non-adherent fraction could be induced to lytic activity against EC, whereas the adherent cells could not. When PBMC were separated, using 2-amino-ethylisothiouronium bromide hydrobromide-treated sheep red blood cells (AET-SRBC), into T- and non-T-cell fractions, the latter fraction could be induced to lyse EC. TCGF-induced cell-mediated EC lysis could not be inhibited using anti-T3 nor antie-LFA-l antibodies. Lysis of EC by TCGF-stimulated effector cells was strongly inhibited by the addition of unlabelled K562 target cells, whereas cold OKT3 hybridoma cells did not exert such an effect. In conclusion: the kinetics of the induction of lytic activity against EC, as well as the cell separation experiments, suggest that short-term-activated NK cells may lyse EC. This hypothesis was confirmed using monoclonal antibody and cold target cell analysis. [ABSTRACT FROM AUTHOR]

Natural killer (NK) activity is inhibited by the contact of peripheral blood lymphocytes with primary monolayer cell cultures of both benign and malignant origin. In this study the effect of interferons (IFNs) on the inhibition has been analysed. Both αIFN- and γIFN-pretreated peripheral blood lymphocytes are effectively inhibited by monolayer target cells. IFN treatment of lymphocytes does not change cytotoxicity against the inhibitory target cells, although it enhances reactivity against NK-sensitive target cells. Treatment of monolayer cells with interferons significantly reduces their inhibitory capacity. However, diminished inhibition of NK activity by the IFN-treated target cells is not associated with increased lysis, probably due to their decreased sensitivity to natural killer cytotoxic factors (NKCF). In 18.5% of the cases studied, monolayer target cells induced endogenous IFN production in lymphocytes. in these cases no inhibition of the NK activity of the effector cells was seen. According to the results of this paper, IFNs have a dual effect on the NK regulatory system: they enhance the NK activity of the effector cells against NK-sensitive target cells, and they change the NK resistant target cells in a way that makes them less inhibitory to NK activity but simultaneously more resistant to the toxic factors secreted by NK cells. [ABSTRACT FROM AUTHOR]

It is now well documented that the mode of primary stimulation in the mouse determines the ratio of Th1-, Th0- or Th2-type T cells obtained. In this paper we determine whether driving and cloning human peripheral blood mononuclear cells (PBMC) non-specifically with interleukin-2 (IL-2) and/ or IL-4 and mitogen, will differentially select T cells for IL-10 production. The presence of IL-2 during culture (with or without IL-4) yielded predominantly Th1-type clones (81% of clones with IL-2 alone and 77% with IL-2 + IL-4) and a low frequency of Th0-type clones (19% of clones with IL-2 and 10% of clones with IL-2 + IL-4), whereas IL-4 alone increased the yield of the IL-4 producing Th0 (35%) clones. The proportions of IL-2-producing clones did not alter with treatment; however, the combination of IL-2 + IL-4 altered the proportions of IL-10-producing clones. 47% of IL-2-cultured cells and 51% of IL-4-cultured cells produced IL-10 respectively, whereas only 22% of clones driven with IL-2 + IL-4 produced detectable levels of IL-10. Thus in the preliminary experiments described here we have demonstrated that the cytokines used to initially drive out human T cells and maintain the T-cell growth can skew the Th1/Th2/Th0 cytokine profiles of the clones obtained from mitogen-stimulated cultures. Moreover and unexpectedly the proportion of IL-10-producing cells is altered. These results are important in interpreting analysis of the cytokine profiles of human T cells and raise questions concerning how we should derive T-cell clones for a cytokine analysis that truly reflects the in vivo situation. [ABSTRACT FROM AUTHOR]

This paper describes a model for investigation of the functional implications of B-cell activation for antigen presentation. Mixed lymphocyte cultures were used to assess the ability of freshly isolated B cells, mitogen-activated B cells and Epstein Burr virus (EBV)-transformed B-cell lines to stimulate the activation and proliferation of allogeneic T cells under a variety of experimental conditions. It was found that resting B cells presented antigen poorly, while activated cells were highly immunogenic. Paraformaldehyde fixation completely eliminated antigen presentation by resting B cells, despite constitutive expression of class II MHC antigens. However, fixation had little effect on antigen presentation by activated B cells that expressed B7-1 and B7-2 in addition to class II major histocompatibility complex (MHC) molecules. Arrest of B-cell activation by serial fixation after treatment with F(ab′)2 fragments of goat anti-human IgM produced cells with variable antigen-presenting capacity. Optimal antigen presentation was observed for cells fixed 72hr after the initiation of B-cell activation. Although both BT-1 and B7-2 antigen expression increased after B-cell activation, it was found that the rate of T-cell proliferation correlated most closely with B7-2 expression. Stimulation of T cells by fixed activated B lymphocytes could be blocked by antibodies directed at class II MHC molecules, indicating involvement of the T-cell antigen receptor. In addition, T-cell proliferation was inhibited by antibodies specific for B7-1 and B7-2 and by the fusion protein CTLA4-Ig, demonstrating a requirement for CD28 signal transduction. The sole requirement of B7 family expression for antigen presentation by B lymphocytes was shown by demonstration of T-cell stimulation by fixed resting B cells in the presence of CD28 antibody as a source of artificial costimulation. [ABSTRACT FROM AUTHOR]

Previously we have identified an immunodominant, eight-residue, epitope core sequence (TAAGNVNI) from the 19 000 MW protein of Mycobacterium tuberculosis, which is recognized in the context of multiple H-2 I-A molecules. In this study, the role of residues flanking this T-cell epitope core was examined, using a series of 20 mer analogue peptides in which the native flanking residues were progressively replaced with L-alanine. Analogue peptides were tested for their capacity to stimulate a CD4+ 19000 MW protein-specific T-cell line, revealing that all but one N-terminal flanking residue could be replaced collectively by alanine without significant loss of stimulatory activity. However, clear H-2-associated differences in the requirement for flanking residues were demonstrated with peptide-specific T-cell hybridomas. In particular, H-2d-derived hybridomas were much more stringent in their requirement for flanking residues than were hybridomas. All polyalanine-substituted peptides bound I-Ab molecules, with affinities similar to the native unsubstituted peptide. In contrast, significantly reduced binding to I-Ad was observed with several analogue peptides, although without a clear relationship to the degree of substitution. Furthermore, in H-2b mice, neither immunogenicity nor cross-reactivity with the native peptide showed a clear inverse relationship with respect to the degree of alanine substitution. The results presented in this paper indicate that flanking residues can influence T-cell specificity and that these effects may be controlled by major histocompatibility complex (MHC) haplotype. [ABSTRACT FROM AUTHOR]

T cells involved in specific and nonspecific co-operation belong to a long-lived population. However, under conditions of limiting dilution of T cells it is not a common event to detect both types of co-operative event in a single microculture well. Likewise, as far as specific co-operation is concerned, the simultaneous expression of both direct and indirect PFC responses in a single microculture well is also unusual. Estimates obtained of the number of PFC generated by one T cell in specific co-operation correspond well to those generated by a single B cell; and it would seem that under conditions of the microculture system there is a marked restriction in the number of B cells that a single T cell may interact with in the short term. This restriction seems to be of a one T-cell to one B-cell relationship. [ABSTRACT FROM AUTHOR]

Antigen-dependent migration inhibition factor produced in cultures of mouse lymph node lymphocytes was further characterized. It was found to bind to Concanavalin A and could be eluted by the appropriate sugar. The pi of the activity was found to be between 6-4 and 6.5 by isoelectric focusing. [ABSTRACT FROM AUTHOR]

Supernatants were collected from keyhole limpet haemocyanin (KLH) primed spleen cells which had been incubated in tissue culture medium with their priming antigen KLH. These non-specific factor (NSF) containing supernatants were then tested in a microculture system for their ability to facilitate an anti-SRBC response of nu/nu or AT x BM spleen cells. It was concluded that: (a) NSF was able to engage a large number of the available pool of sheep erythrocyte (SRBC) specific B cells into an antibody response; (b) this response was characterized by the development of clones expressing plaque-forming cells (PFC), the number of PFC produced within a clone being dependent upon the amount of NSF available in that culture; (c) NSF probably acted directly on B cells, and not via other accessory cell types. [ABSTRACT FROM AUTHOR]

Summary: Microglial inflammation driven by the NACHT, LRR and PYD domain‐containing protein 3 (NLRP3) inflammasome contributes to brain disease and is a therapeutic target. Most mechanistic studies on NLRP3 activation use two‐dimensional pure microglial cell culture systems. Here we studied the activation of the NLRP3 inflammasome in organotypic hippocampal slices, which allowed us to investigate microglial NLRP3 activation in a three‐dimensional, complex tissue architecture. Toll‐like receptor 2 and 4 activation primed microglial inflammasome responses in hippocampal slices by increasing NLRP3 and interleukin‐1β expression. Nigericin‐induced NLRP3 inflammasome activation was dynamically visualized in microglia through ASC speck formation. Downstream caspase‐1 activation, gasdermin D cleavage, pyroptotic cell death and interleukin‐1β release were also detected, and these findings were consistent when using different NLRP3 stimuli such as ATP and imiquimod. NLRP3 inflammasome pathway inhibitors were effective in organotypic hippocampal slices. Hence, we have highlighted organotypic hippocampal slice culture as a valuable ex vivo tool to allow the future study of NLRP3 inflammasomes in a representative tissue section, aiding the discovery of further mechanistic insights and drug development. [ABSTRACT FROM AUTHOR]

Summary: Monocyte‐derived macrophages (MDMs) generated from peripheral blood monocytes are widely used to model human macrophages for in vitro studies. However, the possible impact of different isolation methods on the resulting MDM phenotype is poorly described. We aimed to investigate the effects of three commonly used monocyte isolation techniques on the resulting MDM phenotype. Plastic adhesion, negative selection, and CD14pos selection were compared. Monocyte‐derived macrophages were generated by 5‐day culture with macrophage and granulocyte–macrophage colony‐stimulating factors. We investigated monocyte and MDM yields, purity, viability, and cell phenotype. CD14pos selection resulted in highest monocyte yield (19·8 × 106 cells, equivalent to 70% of total) and purity (98·7%), compared with negative selection (17·7 × 106 cells, 61% of total, 85·0% purity), and plastic adhesion (6·1 × 106 cells, 12·9% of total, 44·2% purity). Negatively selected monocytes were highly contaminated with platelets. Expression of CD163 and CD14 were significantly lower on CD14pos selection and plastic adhesion monocytes, compared with untouched peripheral blood mononuclear cells. After maturation, CD14pos selection also resulted in the highest MDM purity (98·2%) compared with negative selection (94·5%) and plastic adhesion (66·1%). Furthermore, MDMs from plastic adhesion were M1‐skewed (CD80high HLA‐DRhigh CD163low), whereas negative selection MDMs were M2‐skewed (CD80low HLA‐DRlow CD163high). Choice of monocyte isolation method not only significantly affects yield and purity, but also impacts resulting phenotype of cultured MDMs. These differences may partly be explained by the presence of contaminating cells when using plastic adherence or negative selection. Careful considerations of monocyte isolation methods are important for designing in vitro assays on MDMs. [ABSTRACT FROM AUTHOR]

Mast cells are proposed to be one of the targets for mucosal vaccine adjuvants. We previously demonstrated that mucosal adjuvants containing IgG immune complexes could activate connective tissue mast cells enhancing immune responses. Here we suggest that mucosal mast cells (MMC) may also contribute to augmentation of antigen-specific immune responses following treatment with antigens complexed with IgG. We demonstrated that both bone marrow-derived cultured MMC and tissue resident MMC incorporated ovalbumin (OVA) at a greater level in the presence of anti-OVA IgG. Co-culture of OVA/IgG-pulsed bone marrowderived MMC with splenocytes from OT-II mice promoted OVA-specific activation and proliferation of T cells, a process known as cross-presentation. Furthermore, bone marrow-derived cultured MMC underwent apoptosis following treatment with IgG immune complexes, a feature that has been described as favouring phagocytosis of mast cells by professional antigen-presenting cells. [ABSTRACT FROM AUTHOR]

The precise function of tissue factor ( TF) expressed by dendritic cells ( DC) is uncertain. As well as initiating thrombin generation it can signal through protease-activated receptor 2 ( PAR-2) when complexed with factor VIIa. We investigated the expression and function of TF on mouse bone marrow ( BM) -derived DC; 20% of BM-derived DC expressed TF, which did not vary after incubation with lipopolysaccharide ( LPS) or dexamethasone ( DEX). However, the pro-coagulant activity of DEX-treated DC in recalcified plasma was 30-fold less than LPS-treated DC. In antigen-specific and allogeneic T-cell culture experiments, the TF on DEX-treated DC provided a signal through PAR-2, which contributed to the reduced ability of these cells to stimulate CD4+ T-cell proliferation and cytokine production. In vivo, an inhibitory anti- TF antibody and a PAR-2 antagonist enhanced antigen-specific priming in two models where antigen was given without adjuvant, with an effect approximately 50% that seen with LPS, suggesting that a similar mechanism was operational physiologically. These data suggest a novel TF and PAR-2-dependent mechanism on DEX- DC in vitro and unprimed DC in vivo that contributes to the low immunogenicity of these cells. Targeting this pathway has the potential to influence antigen-specific CD4+ T-cell activation. [ABSTRACT FROM AUTHOR]

Based upon the recognition of antiviral compounds and single stranded viral RNA the Toll-like receptors TLR7 and TLR8 are suggested to play a significant role in initiating antiviral immune responses. Here we report the molecular characterization of the chicken TLR7/8 loci which revealed an intact TLR7 gene and fragments of a TLR8-like gene with a 6-kilobase insertion containing chicken repeat 1 (CR1) retroviral-like insertion elements. The chicken TLR7 gene encodes a 1047-amino-acid protein with 62% identity to human TLR7 and a conserved pattern of predicted leucine-rich repeats. Highest levels of chicken TLR7 mRNA were detected in immune-related tissues and cells, especially the spleen, caecal, tonsil and splenic B cells. Alternative spliced forms of TLR7 mRNA were identified in chicken, mouse and human and expressed in similar tissues and cell types to the major form of chicken TLR7. The chicken TLR7+ HD11 cell line and fresh splenocytes produced elevated levels of interleukin-1β (IL-1β) mRNA after exposure to the agonists R848 and loxoribine. Interestingly, none of the TLR7 agonists stimulated increased type I interferon (IFN) mRNA whereas poly(I:C) (a TLR3 agonist) up-regulated both chicken IFN-α and chicken IFN-β mRNA. In contrast, TLR7 agonists, particularly R848 and poly(U) stimulated up-regulation of chicken IL-1β, and chicken IL-8 mRNAs more effectively than poly(I:C). Stimulation of chicken TLR7 with R848 was chloroquine sensitive, suggesting signalling within an endosomal compartment, as for mammalian TLR7. The deletion of TLR8 in galliforms, accompanied with the differential response after exposure to TLR7 agonists, offers insight into the evolution of vertebrate TLR function. [ABSTRACT FROM AUTHOR]

We investigated the protein-binding sites in a DNAse I hypersensitive site associated withbcl-2 gene expression in human B cells. We mapped this hypersensitive site to the coding sequence of exon 2 of thebcl-2 gene in the bcl-2-expressing REH B-cell line. Electrophoretic mobility shift assays (EMSAs) with extracts from REH cells revealed three previously unrecognized B-Myb-binding sites in this sequence. The protein was identified as B-Myb by using a specific antibody and EMSAs. Accordingly, the levels of B-Myb and bcl-2 proteins, and of Myb EMSA activity, were correlated over a wide range of cell lines, representing different stages of B-cell development. Transfection of REH cells with antisense B-mybdown-regulated EMSA activity and the level of bcl-2, and led to the apoptosis of REH cells. Transfection of the bcl-2-non-expressing RPMI 8226 cell line with a B-Myb expression vector induced B-Myb EMSA activity and the expression of bcl-2. Reporter assays indicated that the HSS8 sequence containing the three B-Myb sites may act as an enhancer when it is linked to thebcl-2 gene promoter. Interaction of B-Myb with HSS8 may enhancebcl-2 gene expression by co-operating with positive regulatory elements (e.g. previously identified B-Myb response elements) or silencing negative response elements in thebcl-2 gene promoter. [ABSTRACT FROM AUTHOR]

Long-term cultured CD4+ or CD8+ bovine T-cell lines and clones were established. The CD8+ T-cell fine and clones had a strong lectin-dependent cytotoxity, whereas the CD+ T-cell line did not. Both phenotype cell lines grew in an interleukin-2 (IL-2)-dependent manner and expressed 50,000–55,000 MW and 65,000–75,000 MW proteins associated with a putative IL-2 receptor (IL-2R), as demonstrated by the cross-linking of radioiodinated recombinant human IL-2 (rhIL-2). Additional molecules of 13,000 and 27,000 MW were also observed on CD8+ T cells. The binding of rhIL-2 was blocked by crude bovine IL-2, and Scatchard plot analysis of the binding data showed that both phenotype cells expressed two different affinity IL-2R that had equilibrium dissociation constants of 12–20 pM (3000–6000 sites/cell) and 146–490 pM (16,000–25,000 sites/well). Only IL-2 stimulated DNA synthesis in these cell lines, whereas mitochondrial enzymes activity, protein synthesis and protein secretion were enhanced by IL-2, mitogens and phorbol myristate acetate. The supernatant from mitogen-stimulated CD4+ cells was unable to enhance the DNA synthesis of either the CD4+ or CD8+ lines, whereas both freshly prepared Con A blasts and anti-immunoglobulin-treated bovine B cells showed elevated DNA synthesis under the same conditions. [ABSTRACT FROM AUTHOR]

We raised anti-VH315 antibodies by immunization of rabbits with VH315 fragments, the variable portions of the immunoglobulin heavy chains of MOPC315 myeloma protein. Inhibition radioimmu-noassay using various immunoglobulins as inhibitors showed that the anti-VH315 antibodies specifically reacted with the variable portions of the heavy chains of MOPC315 myeloma protein. When the variable region (VH) gene of the heavy chains was cloned and sequenced from the cells producing the heavy chains detected by the anti-VH315 antibodies, the VH gene was closely related (82% homology at amino acid level) to the VH gene of MOPC315, When we examined the frequency with which the variable region(s) detected by the anti-VH315 antibodies were expressed in eight Ig- null Abelson virus-transformed cell lines (DJ/DJ or VDJ-/DJ). which were able to generate functional V regions during culture, only one cell line, ATB-1, produced a small number of intracytoplasmic/μ-positive cells (VH315+ cells) stained by the anti-VH315 antibodies. The percentage of the total number of the VH315+ cells to the total number of intracytoplasmic μ-positive cells was 0·91% in AT8-1. In the remaining seven cell lines, no VH315+ cells were detected. In the present study we estimate, for the first time at the individual cell level, the frequency of the utilization of the heavy chain variable region(s) identical or closely related to that of MOPC315 in the functional V region formation during early B-cell development. [ABSTRACT FROM AUTHOR]

It has been reported that rare colonies of mast cells may be identified when human bone marrow cells are cultured in agar. Based on this observation, we attempted to culture mast cells from human bone marrow using a modified agar interphase culture. Metachromatically staining cells appearing in culture peaked in number by 2-3 weeks and consisted of basophil-like cells, mast-like cells and larger, granulated cells superficially resembling mast cells. The large cells, which constituted the majority of metachromatic cells, were berberine sulphate-positive but negative for histamine, chloroacetate esterase, and mast cell tryptase. These larger granulated cells were subsequently identified as macrophages by FACS analysis with Leu M3 and by electron microscopy. Berberine sulphatestaining of macrophages was not due to the de novo synthesis of heparin, as shown by analysis of radiolabelled proteoglycans from cultured cells. The macrophage metachromasia was eliminated with the use of agarose. The metachromatic and berberine sulphate-staining of cultured human macrophages was therefore considered to be due to phagocytosed altar. Recognition of this phenomenon will aid in the proper identification of human mast cells and basophils in cultures where agar or similar substances are present, and demonstrates that berberbine sulphate-staining is not specific for heparin. [ABSTRACT FROM AUTHOR]

Fresh lymph-borne (veiled) dendritic cells (L-DC) in the rat are almost totally negative for the interleukin-2 (IL-2) receptor detected by the monoclonal antibody (mAb) MRC OX39. After 16 hr culture more than 90% of L-DC are OX39 positive, and increased levels of expression can be seen within 5 hr culture. In cultures of L-DC and allogeneic lymphocytes, L-DC appear to express the IL-2 receptor more rapidly than lymphocytes. The intensity of labelling of L-DC is variable but maximal levels are similar to those seen on lymphoblasts. Culture in the presence of concanavalin A (Con A)-stimulated spleen cell supernatants or recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) results in a more rapid and intense expression of the IL-2 receptor by L-DC. L-DC cultured following rigorous T-cell depletion, or derived from athymic rats also express the IL-2 receptor after culture with GM-CSF. Cultured, but not fresh, L-DC bind iodinated recombinant IL-2 in a dose-dependent manner and binding is inhibited by excess unlabelled ligand. The amount of IL-2 bound varies but maximal amounts are similar to those bound by lymphoblasts. Following intravenous endotoxin injection, a large proportion of freshly collected L-DC express the IL-2 receptor and the number of L-DC released into the lymph is increased. An antibody to the IL-2 receptor which blocks an allogeneic MLR has no effect on a xenogeneic MLR using rat L-DC as stimulators and mouse lymphocytes as responders. [ABSTRACT FROM AUTHOR]

Lymph-borne dendritic cells (L-DC) collected from the thoracic duct of rats following mesenteric lymphadenectomy are derived from the small intestine. We have cultured these cells in vitro and examined their survival and phenotypic and functional changes. L-DC survive poorly in culture in normal media (< 50% overnight) but survival can be markedly increased by supplementation with Con A-stimulated spleen cell supernatant or recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) but not by recombinant IL-1, IL-2, IFN-γ or by an IL-3-rich supernatant. The effects of GM-CSF are blocked by a specific antiserum. L-DC display heterogeneity for some surface markers, cytoplasmic inclusions and enzyme reactivity. After 16-48 hr culture the pattern of expression is markedly different. The numbers of Thy-1-positive L-DC and the amount of Thy-1 expressed increases, as do the numbers of L-DC expressing OX48 antigen. All L-DC remain Ia positive, but the proportion expressing the iC'3b receptor, non-specific esterase or cytoplasmic DNA inclusions decreases to almost zero. In contrast to Langerhans' cells, fresh L-DC are potent stimulators of an allogeneic mixed leucocyte reaction (MLR) but their potency is considerably increased after 16 hr culture. Also in contrast to Langerhans' cells, the increase in potency is not affected by culture with CAS and is thus unlikely to be dependent on GM-CSF. The changes described in L-DC properties could be related to their role as antigen-presenting cells. [ABSTRACT FROM AUTHOR]

Unstimulated human peripheral blood lymphocytes (HPBL) were found to proliferate when cultured in vitro with interleukin-2 (IL-2). In bulk long-term cultures of HPBL cultured with IL-2, cell numbers usually doubled after 8-11 days of culture, and a 10-fold increase in cell number occurred between the second and third weeks of culture. These cells retained their ability to respond to a panel of T-cell dependent antigens, phytomitogens and allogeneic cells up to Day 21 of culture. The proliferating cells predominantly expressed the T-cell antigens (CD3, CD4 and CD8), but not antigens of natural killer (NK) cells, B cells or mononuclear phagocytic cells. The proportion of cells expressing CD3 and CD4 antigens progressively increased with length of culture. Purified lymphocytes expressing either CD4 or CD8 antigens were also found to be capable of showing a proliferative response to IL-2, especially when provided with autologous accessory cells. However, purified human peripheral blood B cells expressing the Leu 12 antigen did not respond with or without autologous accessory cells. Unlike the responses to phytomitogen, soluble antigens or allogeneic cells, the proliferative responses of HPBL to IL-2 were not inhibited by a monoclonal antibody (OK-1a-1) to the non-polymorphic part of human class II histocompatibility antigens. [ABSTRACT FROM AUTHOR]

Two distinct murine B-cell differentiation factors, designated B 151 -TRY 1 and B 151-TRF2, were described originally as B151 K12 T-cell hybridoma-derived lymphokines that induce immunoglobulin (Ig) secretion by antigen-activated B cells and unstimulated B cells, respectively. In the present study, we found that a highly purified B1 51-TRF1 fraction prepared by reversed-phase high-performance liquid chromatography (RP-H PLC) also has the ability to cause a polyclonal differentiation of unstimulated B cells into IgM-secreting cells in the apparent absence of co- stimulant. The activity of the B151-TRF1 fraction but not the B151-TRF2 fraction on unstimulated B cells was markedly inhibited by addition of a monoclonal antibody (mAb) specific for the B151- TRF 1/IL-S to the culture. To determine whether 8151 -TRF 1/IL-S and 8151 -TRF2 act on distinct populations among unstimulated B cells, the responsiveness of neonatal B cells and adult B cells that had been fractionated by Percoll density gradient centrifugation was assessed. B15 l-TRFI/IL-5 predominantly acted on lower density B cells, which appeared around 3 weeks after birth in the spleen. In contrast, B151-TRF2 could activate both lower and higher density B cells almost equally and B 151 -TRF2-responsive B cells were already present by 1 week of age. Thus, these results suggest that B151-TRFI/IL-5 and B151-TRF2 act on distinct subpopulations among antigen-unprimed normal B cells to induce IgM-secreting cells. [ABSTRACT FROM AUTHOR]

Rat mast cells with the properties of mucosal mast cells (MMC) proliferate in cultures of haemopoietic tissue in the presence of conditioned medium (CM) from antigen- or mitogen-activated T lymphocytes. The present study shows that recombinant rat interleukin-3 (rIL-3) both stimulated the development of MMC from bone marrow (BM) precursors and maintained the proliferation of rat MMC lines in an identical manner to that of CM. The content per cell of the MMC granule- specific proteinase RMCPII was similar in both IL-3- and CM-stimulated cultures. Passage of CM through DEAE-cellulose separated two active peaks that stimulated autologous MMC proliferation. The biochemical properties of peak 1 were similar to those of murine IL-3 and stimulated multi-potential stem cell development in soft agar cultures of SM cells from rats treated with 5- fluorouracil (which enriches for haemopoietic stem cells). RIL-3 was also active in this assay whereas peak 2 was not, demonstrating that peak I contained IL-3 activity. The presence of MMC in the majority of multi-potential colonies in the soft agar cultures confirmed the early stem cell origin of the MMC lineage. The cultured SM-derived mast cells in the rat are analogues of the MMC subset that is most readily observed proliferating in the gastrointestinal tract in response to helminth parasite infection. The demonstration that IL-3 is responsible for the development and proliferation of MMC should lead to a better understanding of the functional roles of these cells. [ABSTRACT FROM AUTHOR]

Ovine mast cells generated in vitro are morphologically and biochemically similar to mast cells localized to the gastrointestinal tract in vivo. Bone marrow (BM)-derived haemopoietic precursor cells were cultured in the presence of conditioned medium (CM) prepared from in vitro-activated lymphocytes from nematode-infected sheep. Mast cells developed rapidly after 2 weeks of culture and could persist for 2-3 months. The presence of sheep mast cell proteinase (SMCP) in the cultured cells correlated with the number of morphologically recognizable mast cells by Leishman-Giemsa staining and is definitive as, in vivo, this enzyme is unique to mast cells. The content of SMCP per cell increased as the cultures developed. In vivo, increased serum concentrations of SMCP are associated with mast cell function at the site of nematode infection. The availability of the cultured cells should now facilitate mast cell biochemical and functional studies. [ABSTRACT FROM AUTHOR]

The macrophage natural resistance gene. Lab, regulates the ability of a selective population of tissue macrophages to control intracellular multiplication of Leishmania donovani by a T-cell independent mechanism. We show here, using mice congenic for Lsh, that this gene also contributes to the acquisition of T-cell-mediated immunity. Whereas both resistant and susceptible mice generate equivalent primary Tall responses to infection, resistant mice show a rapid increase in accessory cell activity, allowing for greater subsequent Tall expansion. This change in accessory cell function correlates with increased class II antigen expression relative to susceptible mice, both in vino during early infection and in vitro in response to induction by interferon-gamma (IFN-γ). Differences in vitro were independent of, but differentially affected by, amastigote infection. [ABSTRACT FROM AUTHOR]

We have analysed homotypic adhesion induced by the phorbol ester TPA in EBV-immortalized Down's syndrome (trisomy 21) and normal lymphoblastold cell lines. Our results show that the trisomy 21 cells aggregated more readily than normal cells. The aggregation of the two types of cell was blocked effectively by the addition of monoclonal antibodies to the α and β chains (CD11a, CD18) of lymphocyte function-associated antigen (LFA-), but not by monoclonal antibodies to LFA-3 and the α chain of p150,95 (CD11c). Since TPA did not increase the expression of CD18 on trisoiny 21 cells, we conclude that the increased adhesiveness is the result of over-expression of CD18 as a result of gene dosage. [ABSTRACT FROM AUTHOR]

Mononuclear cells play an important role in the elimination of immune complexes (IC). In the presence of complement (C) the binding and degradation of IC by mononuclear cells is enhanced at least two-fold. The enhancement of binding is caused by a synergistic interaction of the IC with cellular Fc and complement receptors (R). In the present study we have investigated the contribution of the complement receptors CRI and CR3 of human monocyte cell line U937 on the complement- mediated binding and degradation of immune complexes and soluble aggregates of IgG (AIgG) bearing C3b or iC3b. It was found that deposition of C3b on AIgG enhanced the binding of AIgO to U937 cells at least two-fold. The C3b-mediated enhancement of binding was abolished by anti-CR1. iC3b-bound to AIgG also enhanced the binding of AIgO to the cells. This binding was only partially reduced by anti-CR3 antibodies, but the combination of anti-CR1 and anti-CR3 fully abolished the iC3b-mediated enhancement of binding. These results suggest that both CR1 and CR3 contribute to the complement-mediated binding and degradation of soluble IC by mononuclear phagocytes. [ABSTRACT FROM AUTHOR]

Using a cell culture system, age-associated changes in immune activities were investigated in newly developed, short-lived mouse strains. These SAM-P strains of mice (H-2k), which have a remarkably short life span (around 9 months) under conventional breeding conditions, showed an age-associated early decline in several immune functions, as compared to ordinary strains of AKR/J (H-2k) and C3H/He (H-2k) mice. Their antibody-forming capacity to T-independent antigen, DNP-Ficoll, and natural killer (NK) cell activity showed a markedly early onset of regression and a sharp decline from the level of control mice at 2 months of age. SAM-P strains of mice have a profound defect in antibody response to a T-dependent (TD) antigen, such as sheep red blood cells (SRBC), thus there was only a feeble antibody response to SRBC as early as the age of 2 months, and a negligible response at a later age. In contrast, the allo-specific cytotoxic T lymphocyte (CTL) response of the mice was as high as that of control mouse strains at 2 months of age and declined little until at least 6 months of age. The early age-related functional decline in the immune system of SAM-P mice suggests that these new inbred strains are appropriate models for investigating the age-related appearance of immune dysfunctions. [ABSTRACT FROM AUTHOR]

The effects on lymphokine production of two agents known to potentiate delayed-type hypersensitivity (DTH), pertussigen (pertussis toxin) (PT) and cyclophosphamide (CY) have been investigated. These two agents were administered to immunized mice. Subsequently, lymph nodes and spleen cells were exposed to specific antigen in vitro. The resulting culture supernatants were assayed for the presence of lymphokines. Only supernatants of cells from the mice given PT contained appreciable quantities of interferon-γ (IFN-γ) and stimulated cells of the monocyte-like WEHI-265 cell line to produce procoagulant activator and plasminogen activator. On the other hand, CY was more effective than PT on the production of interleukin-3 (IL-3). Both adjuvants had small enhancing effects on the production of interleukin-2 (IL-2). With either adjuvant, the cell populations induced had a similarly enhanced capacity to transfer DTH. These results demonstrate that the capacity of cells to transfer DTH does not necessarily correlate with their release of particular lymphokines. The potentiation of DTH by cyclophosphamide did not depend on significantly enhanced generation of IFN-γ, procoagulant activator, or plasminogen activator. The amount of IFN-γ in the culture supernatants correlated with their capacity to produce procoagulant activator and plasminogen activator, whereas the amount of IL-2 and IL-3 did not. [ABSTRACT FROM AUTHOR]

The capacity of cloned murine Ia-positive BK-BI-2.6.C6 T cells to present protein antigens to antigen-reactive long-term cultured T-cell lines was investigated. Antigen recognition by T-line cells on presenting BK-BI-2.6.C6 T-accessory cells resulted in efficient production of lymphokines. While antigen-dependent T cells with transient interleukin-2 receptor (IL-2R) expression were not induced to proliferate, T cells with constitutive IL-2R expression proliferated in response to the secreted IL-2. Although antigen presentation by BK-BI-2.6.C6 T cells resulted in a slight induction of IL-2R expression on responding T cells, as measured by flow cytometry, this augmentation was much smaller than that induced by antigen-presenting spleen cells. Thus the inability of antigen-presenting T-accessory cells to stimulate proliferation of responding T cells with transient IL-2R expression appears to reflect a lack of signal(s) necessary for the induction of IL-2R up to a level critical for initiation of cell division. This test system represents an ideal model to investigate the nature of signals required, in addition to triggering of the T-cell antigen receptor, for the induction of IL-2R. [ABSTRACT FROM AUTHOR]

The mechanism by which a Db-specific helper clone (clone 9) and a hybridoma-derived Db-especific helper factor, referred to as ASHF, induced cytotoxic T lymphocyte (CTL) responses to alloantigens was investigated. Clone 9 or ASHF helped CBA thymocytes to produce CTL against B6 (H-2b), but not D2 (H-2d), alloantigens. However, when BDF1 (H-b,d) spleen cells or an equal mixture of B6 or D2 spleen cells were used as stimulator cells, CTL responses to both B6 and D2 were induced. This suggested that clone 9 cells or ASHF could induce the production of long-ranged, non-antigen-specific helper factor(s) in B6-stimulated cultures. One of these long-ranged factors produced in B6-stimulated cultures was found to be interleukin-2 (IL-2). Thus, clone 9, which is a non-IL-2 producer, increased the production of IL-2 by irradiated B6 spleen cells and by CBA anti-B6 cultures by 4·7- and 5·7-fold, respectively. ASHF did not increase the amount of IL-2 produced by irradiated B6 spleen cells but increased the amount of IL-2 produced in CBA anti-B6 cultures by 3·8-fold. Clone 9 cells or ASHF did not increase IL-2 production in D2-stimulated cultures. Upon stimulation with concanavalin A or antigen, clone 9 cells also produced a non-antigen-specific helper factor. This factor (IL-X) synergized with excess human recombinant IL.2 (rIL-2) in the polyclonal activation of BDF1 or D2 CTL precursors. A model that involves the participation of ASHF, clone 9 cells, IL-2 and IL-X in the induction of a cytotoxic response is proposed. [ABSTRACT FROM AUTHOR]

A T helper clone (clone 9), isolated from a H-2d anti-H-2b mixed lymphocyte culture, was previously found to produce an antigen-specific helper factor (ASHF) that could be specifically absorbed out with BIO.A(2R) (KkAkEkDb), but not B10.A (KkAkEkDd), spleen cells. In order to characterize this ASHF further, we have constructed T-cell hybridoma lines by fusing clone 9 cells with the AKR thymoma, 8W5147. One of these hybridoma clones, referred to as clone 25, produced an ASH F that was specific for the Db alloantigen. Immunization of allogeneic C578L/6 mice with clone 9 cells and subsequent fusion of these immune spleen cells with non-secreting myeloma cells led to the isolation of a monoclonal antibody (mAb) (clone 30 IgM) that was capable of neutralizing the helper activity of clone 25 ASHF; Clone 30 IgM affinity column was found to retain clone 25 ASHF; clone 30 IgM column eluates augmented the cytotoxic responses of CBA/J thymocytes to B6 (H-2b), but not D2 (H- 2d), alloantigens. Preabsorption of clone 25 ASHF with Db-bearing spleen cells prior to affinity purification over a clone 30 IgM column resulted in the abrogation of Db-specific helper activity as well as the loss of a 50,000 molecular weight (MW) band in SDS-polyacrylamide gels run under reducing conditions. Clone 25 ASHF was also retained by immunoadsorbents made with an IgG2a mAb (F23.1) the reactivity of which is against the β chain of the T-cell receptor. Furthermore, affinity purification of clone 25 ASHF over a F23.1 affinity column, but not an irrelevant mAb column, also yielded a 50,000 MW molecule. These findings suggest that this particular ASHF may be intimately related to the T-cell antigen receptor. [ABSTRACT FROM AUTHOR]

A limiting dilution (LD) culture system was used to investigate the effect of cyclosporin A (CsA) on the activation and differentiation of human alloreactive cytotoxic T-lymphocyte precursors (CTL-p). CsA reduced in a dose-dependent fashion the frequency of alloantigen-inducible CTL-p. With most normal individuals tested there was a 20- to 50-fold reduction of alloreactive CTL-p frequencies in the presence of 500–1000 ng/ml CsA. Both unseparated T cells and CD8+ T cells were CsA-sensitive under LD culture conditions. Importantly, however, alloreactive CTL-p from two out of 21 normal individuals were found to be largely CsA-resistant. CsA did not affect the growth of MLR-primed CTL in secondary LD culture. Furthermore, CsA slightly inhibited the cytolytic activity of some alloantigen-specific CTL clones. These results arer discussed with respect to the clinical use of CsA in transplantation medicine. [ABSTRACT FROM AUTHOR]

Virtually nothing is known about the basis for the immunosuppression associated with human T. cruzi infection. We have used an in vitro system to explore this effect. Incubation of human peripheral blood mononuclear cells (PBMC) with blood forms of T. cruzi abrogated their responses to suboptimal, optimal and supraoptimal doses of Con A, PHA or PWM, whether or not monocytes were depleted. Killed parasites were not suppressive. Maximal suppression (74%) occurred when the parasites were present during the entire culture period (96 hr), although significant suppression (33%) was seen when the organisms were added 24, 48 or 72 hr after initiation, suggesting that the early stages of lymphocyte activation had been impaired and that a second generation of cells was also affected. The 4-day supernatant medium of a T. cruzi suspension supported PBMC responses to Con A as well as medium incubated alone, indicating that suppression did not result from parasite removal of essential nutrients. Furthermore, 96 hr after mitogenic stimulation, the proportions of viable PBMC in cultures containing or lacking the parasites were comparable. Although T. cruzi binds Con A and PHA, this absorption was not the cause of reduced responsiveness since optimal concentrations of Con A and PHA remained in solution under our conditions. Levels of IL-2 in PHA- stimulated PBMC cultures were markedly reduced in the presence of T. cruzi. However, exogenous IL-2 failed to restore lymphocyte responsiveness. T. cruzi neither absorbed nor inactivated IL-2. Thus, the noted suppression appeared to involve at least deficient production and utilization of IL-2. [ABSTRACT FROM AUTHOR]

Recombinant interleukin-2 (rIL-2) induced highly purified human tonsillar B cells to differentiate into immunoglobulin (Ig)-producing cells in vitro. The B-cell response was not due to rIL-2- contaminating substances, but reflected the activity of IL-2 itself, since it was inhibited by addition to the cultures of anti-TAC monoclonal antibody. The rIL-2-induced B-cell response was apparently not mediated by factors released by residual T cells present in B-cell suspensions at undetectable levels, since supernatants (SN) from unstimulated autologous T cells cultured at concentrations even much higher than those possibly contaminating B-cell suspensions did not induce any detectable Ig production. In addition, the Ig production by B cells cultured with SN prepared from high numbers of autologous T cells stimulated with rIL-2, as well as from allo-activated or mitogen-stimulated T cells, was of the same magnitude as the Ig production resulting from direct addition of rIL-2 concentrations comparable with those present in the supernatants. After centrifugation on Percoll density gradients, most of the tonsillar B cells responsive to rIL-2 were recovered in the lower density cell fraction containing a number of larger activated B cells. Moreover, B-cell enriched suspensions from peripheral blood (PB) (which usually contains a lower number of in vivo activated B cells than tonsil) showed poor or no response to rIL-2 alone, but displayed significant Ig production when rIL-2 was added to the cultures in the presence of Staphylococcus aureus Cowan I (SAC) bacteria. Taken together, these data indicate that IL-2 can directly induce differentiation into Ig-producing cells of in vivo or in vitro activated human B cells. [ABSTRACT FROM AUTHOR]

Primary and secondary bovine allogeneic mixed leucocyte cultures were examined for the generation of antigen-specific cytotoxic leucocytes. While optimal generation of murine and human cytotoxic T lymphocytes typically requires 4-8 days, alloantigen-specific cytotoxic bovine leucocytes were demonstrated consistently only after prolonged incubation periods, optimally found to be about 15 days. Restimulation of long-term bovine mixed leucocyte cultures with the original stimulator population revealed responder cells demonstrating augmented alloantigen-specific lytic activity. When placed into human recombinant interleukin-2, responder cells expanded and required passaging every 3 -4 days. The same was not true of cells placed into interleukin-2-free medium. Cells cultured in interleukin-2-containing medium retained alloantigen specificity after 10 weeks of culture. Moreover, they continued to display total dependence on human, simian or bovine interleukin-2 for growth. [ABSTRACT FROM AUTHOR]

Antigen-specific T-cell activation is mediated via the CD3-T1(antigen receptor) complex. and monoclonal antibodies to both CD3 and T1 cause a rapid rise in intracellular Ca2+. This calcium mobilization is not inhibited by monoclonal antibodies to CD2. The rise in calcium mobilization induced by purified PHA (PHA-P) does not occur in a cell line which lacks CD2 expression, and can be blocked in other I cells by anti-CD2 antibodies. A combination of monoclonal antibodies to different epitopes of CD2 causes calcium mobilization and mitogenesis. Reagent grade PHA (PHA- M) induces calcium moblization in cells that lack CD2, and its effects in other T cells cannot be blocked by anti-CD2 antibodies. The effects of PHA-P and PHA-M arc thus mediated predominantly through different activation pathways. [ABSTRACT FROM AUTHOR]

The requirements for triggering human B cells to DNA synthesis by T-independent polyclonal activators were examined. Optimal S phase entry of purified resting B cells infected with Epstein-Bar virus (EBV) or confronted with killed particles of Staphylococcus aureus Cowan Strain I (SAC) required a high density of cells in culture. Experiments varying culture vessel geometry and culture volumes revealed that the initial limiting quantity was a soluble activity generated in the B-cell cultures. A parallel observation was noted in the requirements for the sustained growth of EBV-transformed lymphoblasts. Autostimulatory soluble factors harvested from such cultures were able to augment DNA synthesis in low density cultures of resting cells triggered by EBV or SAC. Below a critical cell number, however, soluble factors by themselves, were not sufficient either for supporting primary B-cell responses or for maintaining the proliferation of transformed lymphoblasts. By employing conditions which encouraged cell contact it was found that a second, non-harvestable factor requiring cell proximity for its action was also necessary to promote B-cell growth. The implications of these findings for autocrine and paracrine models of B-cell activation are discussed. [ABSTRACT FROM AUTHOR]

Preactivation of ovine peripheral blood and lymph node mononuclear cells with mitogenic doses of concanavalin A (Con A) induced cells which suppressed mitogen-stimulated proliferative responses of untreated autologous and allogeneic responder cells. The degree of suppression varied with preactivating doses of Con A, length of preactivation time, and ratio of preactivated to responder cells. The role of macro- phages in generation of suppressor cells was not evaluated. However, macrophages were not required to mediate suppression in cocultures, as lymphoblasts depleted of macrophage by plastic adherence and nylon wool columns mediated equal, and often greater, suppression than unseparated, preactivated cells. Suppressor cell activity in peripheral blood increased from the neonatal period to adulthood. Supernatants from Con A preactivated cell cultures with detectable interleukin-2 activity abrogated suppression when added at 0 and 24 hr of the 72 hr coculture period, suggesting that Con A-induced suppressor cells exert their function by decreasing available levels of IL-2 in the cocultures. [ABSTRACT FROM AUTHOR]

(1) Oil-elicited guinea-pig peritoneal macrophages (MPs) cultured for 2-3 days in medium containing supernatant of concanavalin A-activated lymphocytes (lymphokine, LK) generated large amounts of hydrogen peroxide (H2O2), as detected by the horseradish peroxidase (HRP)dependent oxidation of phenol red, in the absence of further stimulation. (2) H2)2 production increased with the duration of exposure to LK and was evident at high dilutions of supernatant (1/64). (3) Parallel cultures of MPs in medium or a supernatant of non-activated lymphocytes also increased their H2O2 production during cultural but levels at all time intervals were significantly lower than those measured in LK treated cultures. (4) The marked increase in H2O2 production was associated with only a moderate increment in superoxide (O2-) liberation and this was not specific for LK treated cells. (5) Detection of LK-dependent H2O2 production was dependent on ongoing pinocytosis during the assay. This and other arguments suggest that the HRP-phenol red assay, as applied here, detects H2O2 generation occurring at the level of intracellular vesicles and it is concluded that LK elicits H2O2 production that is limited to the intracellular compartment. (6) H2O2 is, apparently, derived by non-enzymatic dismutation of Of taking place within the cell; (7) LK treatment of MPs also resulted in a significant reduction in catalase activity. [ABSTRACT FROM AUTHOR]

Deoxyguanosine (dGuo) is a purine nucleoside phosphorylase (PNP) sustrate which has been shown to inhibit T lymphoblast growth, PHA-induced cell proliferation and suppressor T cell activity. Low dGuo concentrations (0·5-5 μM) increase interleukin-2 (IL-2) sensitivity of IL-2-dependent cultured T cells (CTC). DGuo alone has no direct mitogenic effect on CTC. The increased IL-2 sensitivity of CTC was more marked in the presence of low IL-2 concentrations than high ones. CTC incubated with dGuo at 37°C for 24 hrs absorbed more exogenous IL-2 than control non-treated CTC. CTC incubated at 4° in the presence of dGuo did not absorb any more IL-2 than control CTC. FACS analysis further showed increased Tac expression on CTC due to dGuo. These findings indicate that a 37° incubation of CTC with dGuo increases the number of CTC IL-2 receptors. dGuo was not found to increase IL-2 production by PHA-stimulated PBM. Therefore, low dGuo concentrations selectively augment the sensitivity of cells responding to IL-2 by increasing IL-2 receptors. [ABSTRACT FROM AUTHOR]

Colonies of cells termed 'giant granular leucocytes' (GUL) displaying natural killer (NK) activity were generated in cell culture. The prominent feature of these cells was the formation of large cytoplasmic pool-the `theca'-filled up with glycogen. This was demonstrated by the strong positive red staining of the theca with periodic acid Schiff reagent (PAS) which was abolished by prior treatment with amylase. Two different procedures were employed for obtaining colonies of NK-GGL. In the first, mice were injected either with killed Corynebacterium parvum or with killed Bordetella pertusis preparations and their mesenteric lymph-node cells were grown on syngeneic X-irradiated embryonic skin fibroblast monolayers. At the foci of GGL formation the fibroblasts were killed and the cleared areas thus formed were populated by adherent GGL. In the second procedure, supernates from rat or mouse spleen cultures stimulated with concanavalin A (Con A)-Interleukin-2 (IL-2)-were added to cultures of spleen and lymphn-ode cells prepared from either ordinary or from athymic nude mice. Richest GGL populations developed when rat IL-2 was added to cells of nude mice. Mouse IL-2 was less consistent. With nude mouse cells it stimulated, either mast cells or GGL, or both; rat IL-2 did not stimulate mast-cell differentiation in nude mouse cultures. In contrast, supernates from lymph-node cell cultures prepared from mice infected with Schistosoma mansoni. Mucosal mast cell-stimulating factor (MMSF) stimulated the formation of colonies of mast cells but not GGL. When MMSF was added as late as 23 days, colonies of young mast cells appeared and mast cells progressively increased in number. When rat IL-2 was added to such mature mast-cell cultures on the 30th day, colonies of cytolytic-GGL appeared. These observations indicate that precursors of mast cells and GGL persist in the cultures and preserve their potential to be stimulated by T-cell factors. GGL-NK cells developed on monolayers prepared from whole embryos released substance that displayed morphology and staining characteristic of mucus. Evidence gathered from in-vitro and in-vivo studies links the in-vitro GGL-NK cells to motile cells that inhabit the mucosal epithelium. Based on the observations, a hypothesis on the function of NK cytotoxicity is brought forward. It proposes the replacement of ordinary epithelial cells, which are killed during a proliferative and differentiative response of other cells at the onset of an infection course. [ABSTRACT FROM AUTHOR]

The syngeneic mixed lymphocyte reaction (SMLR) is the proliferative response of T lymphocytes cultured with syngeneic non-T lymphocytes. In previous studies, we found that the SMLR reaches adult level of activity at 4 weeks of age in BALB/c mice. We now report that the SMLR declines with age in this strain. The decline was first documented at 12 months of age, when non-T spleen cells were less able to stimulate young adult T cells than were non-T cells from 2 to 3 month-old mice. Splenic T cells from 12 month old mice were as responsive as splenic T cells from 2 to 3 month old mice. By 24 months of age, mice had no significant SMLR activity. Splenic T cells from 24 month old mice did not respond and splenic non-T cells did not stimulate SMLR when cultured with cells from young adult mice. Finally, suppressor cells were demonstrated in spleen cell preparations from 24 month old mice and may explain or contribute to the impaired SMLR in these animals. [ABSTRACT FROM AUTHOR]

The results presented here show that concanvalin A (Con A) stimulated mouse spleen lymphocytes cultured at 1/10th optimal concentration incorporated less [³H]-TdR than those cultured at the optimal concentration. It was possible to increase the response of 1/10th optimal concentration lymphocytes by supplementing the cultures with macrophage-rich peritoneal exudate cell (PEC) preparations. This effect was dependent on the PEC concentration. [³H]-TdR incorporation by lymphocytes at optimal concentration was inhibited by the addition of PEC to the cultures. Thymocyte cultures at 1/10th optimal concentration also showed increased [³H]-TdR incorporation when supplemented with PEC but there was no inhibition effect at optimal concentration. PEC which had been pretreated with fresh mitomycin C to prevent cell division were as effective as controls at promoting the stimulation of 1/10th optimal concentration lymphocytes. PEC which had been killed were much less effective than controls. [ABSTRACT FROM AUTHOR]

Human monocytes incubated in vitro for 20 hr at 37 release a factor which is cytotoxic to a number of human and routine tumour cell lines: untransformed cells appear to be less susceptible. A similar factor is produced under comparable conditions by myelomonocytic leukaemic cells and by macrophages derived from monocytes by in vitro culture for 8 days. Maximum production of the factor occurred in the presence of foetal calf serum or autologous plasma and endotoxin. The factor is newly synthesized in culture as its production is reduced if the monocytes are treated with cycloheximide or actinomycin D or incubated at lower temperatures. Freshly isolated monocytes do not release the factor on freeze-thaw or hypotonic lysis. The monocyte cytotoxin has apparent molecular weights of 34,000 on Ultrogel AcA54 gel filtration and 140,000 on gradient polyacrylamide gel electrophoresis; it has β2γt electrophoretic mobility in polyacrylamide gel and does not appear to be C3a or arginase. [ABSTRACT FROM AUTHOR]

Rabbit spleen and mesenteric lymph node cells were treated with a monoclonal anti-rabbit 1-lymphocyte antibody (MAb) and complement and the effect of the treatment on various lymphocyte functions was determined. Lysis of spleen and mesenteric lymph node cells reactive with this MAb. 9AEIO. essentially eliminated their proliferative responsiveness to allogeneic lymphocytes in the mixed lymphocyte reaction and to the T-cell mitogens, concanavalin A and phytohaemagglutinin; responsiveness to the B-cell mitogen, anti-Immunoglobulin (Ig) was not decreased by lysis of 9AE10+ cells. In addition, the 9AE 10+ cells were found to be necessary for the secondary in vitro antibody response to the T-dependent antigen sheep red blood cells (SR BC), as removal of 9AE l0+ cells blocked the generation of plaque forming cells (PEC) in culture. The PFC's themselves were not sensitive to lysis by 9AE 10 MAb and complement. Thus, the 9AE 10 MAb appears to recognize cells which have functions characteristic of T lymphocytes and this monoclonal antibody will be useful in further studies of the rabbit cellular immune system. [ABSTRACT FROM AUTHOR]

In this study, the mechanism by which Concanavalin A (Con A) pre-treated lymphocytes sup- press mitogen-induced proliferation of responder cells was investigated. Responder-cell proliferation could only be suppressed when these cells were co-cultured with the Con A pre-treated cells but not with their supernatants, nor in chambers where the suppressor cells were separated from the responder cells by a millipore filter. Suppression could not be mediated by heat-killed Con A-activated cells or lysates from these cells. Trypsinization of the Con A-induced suppressor cells resulted in loss of suppressor activity which could be restored if the cells were allowed to recover over- night. Trypsinization of the responder cells, however, before their co-culture with the Con A pre-treated cells did not affect suppression. Addition of cytochalasin B to the co-culture resulted in reduced suppression and cycloheximide treatment of the suppressor cells abolished their activity. The results indicate that for optimal suppression to occur, cell-to-cell contact is required and viable, intact Con A-inducible suppressor cells, actively synthesizing protein are essential. Furthermore, suppression may be mediated via a membrane receptor on the suppressor cell as manipulations of normal membrane function may abolish suppression. [ABSTRACT FROM AUTHOR]