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It has been reported that, in early pregnancy in mice, there is an increase in the number of immunoglobulin-secreting cells in the lymph nodes which drain the uterus. This paper describes the results of further investigations provoked by interest in these early changes. Increases in the numbers of immunoglobulin-secreting cells were observed in syngeneically, but scarcely or not at all in allogeneically, mated mice. Increases were not observed in surgically sterilized female mice inseminated by normal males. However, subcutaneous injection of sperm provoked massive increases in the numbers of immunoglobulin-secreting cells in the lymph nodes draining the injection site. The changes were compared with those provoked by the injection of spleen cells and LPS. The results are discussed in relation to (i) the nature of the interactions provoking the increases in the number of immunoglobulin-secreting cells and their possible relationship to immunosuppression, and (ii) the relative immunological unresponsiveness which the female shows to the challenge of inseminated sperm. [ABSTRACT FROM AUTHOR]

There is considerable confusion over whether the antigen-specific T suppressor factors (TsF) described by different authors are indeed equivalent. This paper investigates whether monoclonal TsF3, obtained from hybridomas derived from mice injected subcutaneously with NP derived spleen cells. is functionally equivalent to the conventional T suppresser factor, produced by mice injected intravenously with chemically reactive, water soluble haptene (picrylsulphonic acid and oxazolone thioglycolic acid). Comparison of monoclonal anti-NP TsF3 with conventional anti-picryl and anti-oxazolone T suppressor factor showed that both armed the non-specific T accepter cell (Tacc) which was sensitive to cyclophosphamide and adult thymectomy. Moreover, non-specific inhibitor (nsINH) of the transfer of contact sensitivity was released when antigen, together with major histocompatibility complex products (MHC). reacted with conventional or monoclonal TsF on the surface of the non-specific T accepter cell. The interaction of monoclonal TsF3 with antigen, which led to the release of nsINH, required the presence of MHC and was I-J restricted. However, there was no Igh-1 restriction. The equivalence of conventional anti-picryl and anti-oxazolone TsF has been demonstrated by arming the Tacc with a mixture of these two suppressor factors, and then triggering the release of nsINH with the mixed haptene ‘picryl-oxazolone-lysine’ which crosslinks separate molecules of TsF. A similar equivalence of conventional anti-oxazolone TsF and monoclonal anti-NP TsF3 was demonstrated using the mixed hapten ‘NP-oxazolone-lysine’ to trigger the release of nsINH. It was concluded that monoclonal TsF3 and conventional TsF were equivalent, and that both had an indirect mode of action through the non-specific T acceptor cell which led to the production of non-specific inhibitor. [ABSTRACT FROM AUTHOR]

This paper uses B-lymphocyte cloning methods to quantify the effects of anti-μ chain antibody on immature and mature B cells. Nude mouse spleen lymphocytes were incubated with various concentrations of sheep anti-mouse μ chain antibody for times varying from 10 min to 24 h. They were then washed and plated in the agar B-cell colony formation assay. Five to six days later, control B cells had developed into colonies with a plating efficiency of about 5%. B cells from newborn mice pretreated with anti-μ yielded fewer colonies. Remarkably low concentrations sufficed to inhibit subsequent mitogenesis. For example, 3 μg/ml acting for 1 h or 0.1 μg/ml acting for 24 h gave > 50% inhibition. Adult B cells were about thirty-fold more resistant to negative signalling. Immature cells became more profoundly inhibited as anti-μ treatment was prolonged. Anti-Ia or anti-H2 antibodies, in the absence of complement, did not deliver a negative signal. Anti-μ pretreatment also reduced the capacity of immature B cells to form clones of anti-hapten antibody-forming cells in a liquid microculture system where the triggering stimulus was a T-cell independent antigen. Mature 'T-independent' B cells were not inhibited. Populations of hapten-specific B cells prepared by the hapten-gelatin method were investigated in the agar cloning system. Pretreatment of immature cells with anti-μ reduced their capacity to form colonies, this subpopulation of cells behaving like unfractionated B cells. Furthermore, hapten--HGG delivered a negative signal also. Mature hapten-specific cells or unfractionated immature spleen cells formed normal numbers of colonies following hapten HGG treatment. Overall, the studies support the view that anti-μ antibody and hapten--HGG deliver strong negative signals to immature but not mature cells with appropriate receptors. The value of anti-μ as a model, universal tolerogen was supported. Fluorescence-activated cell sorter (FACS) analysis was performed to study the relationships between functional inhibition and Ig receptor modulation. We confirmed that the IgM receptors of immature B cells are more readily modulated by anti-μ antibody than those of mature cells. Furthermore, the receptor regeneration could be partially inhibited amongst immature but not mature B cells. There was not a close quantitative relationship between the degree of modulation and the degree of functional inhibition. The results did not support the view that irreversible receptor modulation as such was the cause of functional inhibition. [ABSTRACT FROM AUTHOR]

Macrophages and dendritic cells ( DCs) in murine spleen are essential for the maintenance of immune homeostasis by elimination of blood-borne foreign particles and organisms. It has been reported that splenic DCs, especially CD8 α+ CD103+ DCs, are responsible for tolerance to apoptosis-associated antigens. However, the molecular mechanism by which these DCs maintain immune homeostasis by blood-borne apoptotic cell clearance remains elusive. Here, we found that the CCL22/ CCR4 axis played a critical role in the maintenance of immune homeostasis during apoptotic cell clearance by splenic CD8 α+ CD103+ DCs. The present results revealed that systemic administration of apoptotic cells rapidly induced a large number of CCL22 and CCR4+ regulatory T (Treg) cells in the spleen of C57 BL/6J mice. Further study demonstrated that CD8 α+ CD103+ DCs dominantly produce much higher CCL22 than CD8 α+ CD103− DCs. Moreover, the transient deletion of CD8 α+ CD103+ DCs caused a decrease in CCL22 levels together with CCR4+ Treg cell percentage. Subsequently, the levels of some pro-inflammatory cytokines, such as interleukin-17 and interferon- γ in the spleen with the absence of CD8 α+ CD103+ DCs increased in response to the administration of apoptotic cells. Hence, intravenous injection of apoptotic cells induced a subsequent increase in CCL22 expression and CCR4+ Treg cells, which contribute to the maintenance of immune homeostasis at least partially by splenic CD8 α+ CD103+ DCs. [ABSTRACT FROM AUTHOR]

The precise mechanisms responsible for immunosenescence still remain to be determined, however, considering the evidence that disruption of the organization of primary and secondary lymphoid organs results in immunodeficiency, we propose that this could be involved in the decline of immune responses with age. Therefore, we investigated the integrity of the splenic microarchitecture in mice of increasing age and its reorganization following immune challenge in young and old mice. Several differences in the anatomy of the spleen with age in both the immune and stromal cells were observed. There is an age-related increase in the overall size of the white pulp, which occurs primarily within the T-cell zone and is mirrored by the enlargement of the T-cell stromal area, concurrent to the distinct boundary between T cells and B cells becoming less defined in older mice. In conjunction, there appears to be a loss of marginal zone macrophages, which is accompanied by an accumulation of fibroblasts in the spleens from older animals. Furthermore, whereas the reorganization of the white pulp is resolved after several days following antigenic challenge in young animals, it remains perturbed in older subjects. All these age-related changes within the spleen could potentially contribute to the age-dependent deficiencies in functional immunity. [ABSTRACT FROM AUTHOR]

Follicular dendritic cells (FDC) are important stromal cells within the B-cell follicles and germinal centres (GC) of secondary lymphoid tissues. FDC trap and retain native antigens on their surfaces in the form of immune complexes that they display to B cells, in order to select those cells with the highest antigen affinity. MicroRNAs are short, non-coding RNAs that are approximately 18-25 nucleotides in length that regulate gene expression at the post-transcriptional level by repressing the translation of target genes. In the current study, in vivo and in vitro systems were used to identify microRNAs that were potentially expressed by FDC. Constitutive lymphotoxin- β receptor (LT βR) stimulation is required to maintain FDC in their differentiated state. We show that the rapid de-differentiation of spleen FDC that accompanied LT βR-blockade, coincided with a significant decrease in the expression of mmu-miR-100-5p, mmu-miR-138-5p and mmu-miR-2137. These microRNAs were shown to be expressed in the FDC-like cell line, FL-YB, and specific inhibition of mmu-miR-100-5p significantly enhanced expression of Il6, Ptgs1/2 and Tlr4 mRNA in this cell line. The expression of Il6, Ptgs1/2 and Tlr4 by FDC play important roles in regulating GC size and promoting high-affinity antibody responses, so it is plausible that mmu-miR-100-5p may help to regulate the expression of these genes during GC reactions. [ABSTRACT FROM AUTHOR]

Priming of CBA/J mice with minute doses of protein antigens (Ag) leads to high IgE antibody (Ab) titres in the immune sera of these animals. In contrast priming with large doses elicits only a marginal production of IgE Ab. In vitro restimulation of spleen cells from animals primed with large doses and lacking of vivo IgE Ab leads to a burst of IgE Ab-forming cells. This in vitro ammnestic response is lacking in mice primed with minute doses of Ag. In order to trace the cellular basis of the of vitro IgE memory response we have extended the analysis of the distribution of Ab isotypes to Ag-primed IgG1-deficient Δ5′Sγl mice. The data presented here must be interpreted as followed. Priming of mice with minute doses of Ag leads to a direct switch from IgM to IgE Ab expression in both strains. These animals have high IgE Ab titres without establishing an IgE memory. The direct switch was verified by polymerase chain reaction and Southern blot analysis of switch circle DNA isolated from Ag-specific B cells of CBA/J mice primed with minute doses of Ag. In contrast to immunization with minute doses, priming with large doses of Ag fails to induce in rive IgE Ab production in CBA/J and Δ5′Sγ1 mice but establishes a Bε memory in CBA/J mice which involves IgG1-bearing intermediate B cells. In rive these Bε memory cells do not enter the status of IgE Ab-producing cells. In vitro they can be released from this anergy and presumed suppression and develop in an ammnestic response into a large population of IgE Ab-forming B cells. This increase in the number of IgE Ab-producing cells after restimulation in vitro is lacking in Δ5′Sγ1 mice, apparently because of their inability to generate IgG1-expressing precursor cells. The notion of a sequential switch and an IgG1 intermediate Bε memory status is also supported by depletion and inhibition experiments. Elimination of IgG1-expressing B cells in CBA/J mice primed with high doses of Ag prevents the IgE Ab burst after in vitro challenge with Ag. The data further suggest that the two switch pathways are not mutually exclusive and that the Ag dose can decide which pathway is preferentially used. [ABSTRACT FROM AUTHOR]

Analysis of five monoclonal autoantibodies, rheumatoid factors produced by hybridomas generated from spleen cells of BALB/c mice repeatedly infected with A/PR/8/34 human influenza A virus, revealed that they recognized distinct but spatially related epitopes. The differing isoallotypic specificity of the IsM and IRA monoclonal antibodies correlated with the presence of I1e258 and Ala305, respectively. Although these data suggest that the epitopes recognized are within the CH2 domain, all antibodies failed to inhibit IgG antigen reactivity with Staphylococcus aureus protein A (SpA), C1q, mouse C3, human FcγRI or mouse FcγRII, activities known to be predominantly determined by CH2 domain structures. Reactivity of the IgA antibody, Z34, with IgG2b allowed further specificity studies using a panel of 26 mutant IgG2b proteins, each having single amino acid replacements over the surface of the CH2 domain. The only substitution that affected Z34 reactivity was Asn/Ala297, which destroyed the glycosylation sequon, resulting in secretion of an aglycosylated IgG molecule. The epitope recognized by Z34 therefore seems to be located outside of the FcγR and C1q binding sites, but to be dependent on the presence of carbohydrate for expression. In contrast to the binding studies, complement activation by aggregated IgG2a, through classical or alternative pathways, was inhibited by the presence of autoantibodies. The functional significance of isotype-specific autoantibody in immune regulation is discussed. [ABSTRACT FROM AUTHOR]

Infection in mice with an attenuated strain of Salmonella abortusovis (SAO), a specific pathogen for sheep, was used as a convenient model to understand further the induced immunity against SAO. The hypovirulent Rv6 strain, subcutaneously inoculated in salmonella-susceptible BALB/cby (Ity') mice, colonized the spleen and the liver in less than 6 days post-infection (PI) to be cleared after Day 28 PI. Simultaneously, an increase in spleen cell numbers, splenomegaly and hepatic granulomatous lesions developed to a maximum level on Day 9 PI. In spleen of uninfected mice, the number of Thy-1.2+ cells represents twice the number of surface immunoglobulin-positive cells (sIg+). Cytofluorometric analysis of the spleen lymphoid cell subsets showed a significant increase (10 times, P<0.05)in the number of sIg+ cells from Day 6 to Day 28 PI compared to control values. The number of Thy-1.2+ cells also significantly increased, to a lesser degree than the sIg+ cells, on Day 2 and on Day 16 PI (twice control values, P&lt0.05), but decreased on Day 6 PI compared to Day 2 PI. The highest L3T4+:Lyt-2+ ratio was observed on Day 2 PI and the lowest on Day 9 PI. On Day 28 PI, the number of sIg+ cells was still greater than the number of Thy-l.2+ cells. The granulomatous lesions were observed in the liver as early as Day 2 PI and their frequency was maximal on Day 9 PI. Immunohistochemical analysis of the granulomatous lesions showed that macrophages (F4/80+, Macl+) were the basic cells and that L3T4+ cells were the predominant T cells. In well-developed granulomas observed on Day 9 PI, macrophages were in the centre whereas L3T4+ T cells were preferentially located at the periphery. T cells expressing Lyt-2 antigen were rarely detected. Variations in the proportion of lymphoid cells in the spleen and in hepatic granulomatous lesions suggest different and complementary effector mechanisms in induced immunity against SAO. [ABSTRACT FROM AUTHOR]

Twenty-three monoclonal antibodies (mAbs) against Giardia muris were obtained in two fusions using spleen cells of immunized mice. Similarly, the fusion of mesenteric lymph node (MLN) cells of an infected mouse yielded 15 mAbs. Three mAbs arising from spleen cells and two from MLN (all five were IgM) were able to kill trophozoites in the presence of guinea-pig complement (GPC). They can agglutinate trophozoites and also impair the movement of the flagella in the absence of GPC. I.p. treatment of mice with mAb 45C on Days -1, 1.3 and 5 of infection significantly reduced the intestinal parasite burden. Indirect immunofluorescence assays on live and fixed trophozoites revealed that the cytotoxic mAbs were directed against antigens located on the periphery of the body, the sucking disc, the flagella and the ventro-lateral flange. In Western blots, our mAbs recognized major 36,200 and 30,300 MW glycoproteins located on the surface of the parasite. [ABSTRACT FROM AUTHOR]

In an attempt to identify urodele amphibian lymphocyte subpopulations by their surface markers, we prepared hybridomas from BALB/c mice spleen immunized with axolotl (Ambystoma mexicanum) blood and splenic leucocytes and purified immunoglobulins. Sixty-five hybridomas were selected and subsequently subcloned. Among numerous monoclonal antibodies (mAbs) thus obtained, four mAbs were extensively characterized by immunoblotting, single and double fluorescence and immunohistology. MAb 34.38.6 recognizes polypeptides between 65,000 and 72,000 MW and labels in immunofluorescence nearly all thymocytes, 60-63% splenic lymphocytes of normal animals but only 9% splenic lymphocytes in thymectomized animals. MAb 19.14.2 reacts with a 98,000 MW protein and labels a restricted lymphocyte population in thymus (52-77%) and spleen (20-25%). The immunohistological study demonstrates that 34.38.6 and 19.1 4.2 label most thymocytes and a large proportion of spleen leucocytes including lymphocytes, granulocytes and macrophages. In addition, 19.14.2 labels some large interdigitating cells in thymic epithelial areas and splenic cords. MAbs 33.45.1 and 33.101.2, respectively, recognize heavy (72,000-88,000 MW) and light (20,000-27,000 MW) axolotl immunoglobulin chains. They do not react with thymocytes but label a splenic lymphocyte population not labelled by mAb 34.38.6. The proportion of surface immunoglobuline-positive (sIg+) lymphocytes in spleen is not altered by thymectomy. MAb 33.101.2 labels 40-48% of splenic lymphocytes, 33.45.1 stains only 14% of these same cells. This suggests some interesting heavy-chain isotypic differences in axolotl. For the first time in urodele amphibians, mAbs differentiate T-like and B-like lymphocyte populations by their membrane markers. This will allow further analysis of the axolotl immune system. [ABSTRACT FROM AUTHOR]

Spleen cells from MRL-lpr/lpr, CBA and BALB/c mice were cultured in vitro and assayed for production of anti-nuclear antibodies. Spleen cells from all species produced IgM antibodies to a nRNP (Ul-RNP)-specific antigen and to double-stranded DNA (dsDNA) after stimulation with LPS. The specificity of the anti-nRNP antibodies was shown, by immunoblotting, to be directed against the 33,000 MW polypeptide of nRNP/Sm. CBA mice produced more IgM autoantibody in vitro than MRL/lpr or BALB/c mice. In contrast, IgG anti-nRNP and anti-dsDNA antibody were not produced by any of the strains. Our data show that anti-nRNP and anti-dsDNA precursor B cells are part of the normal murine immune repertoire and are not confined to the MRL/lpr strain. This suggests that the spontaneous development of anti-nRNP and anti-dsDNA antibodies associated with systemic lupus erythematosis (SLE) is dependent on clonal stimulation and removal of suppressive influences. [ABSTRACT FROM AUTHOR]

A limiting-dilution frequency assay was employed to estimate the increased production of cytolytic T lymphocytes (CTL) associated with Corynebacterium parvum-induced regression of the P815 mastocytoma growing subcutaneously in semisyngeneic mice. It was found that intratuinour C. parvum functioned to augment greatly the underlying concomitant production of CTL that occurs normally in response to a progressively growing untreated immunogenic tumour. The lymph node draining a C. parvum-treated tumour contained about eight times more CTL than the lymph node draining a control turnout Intratumour C. parvum also caused a large increase in CTL production in the spleen and an increase in the number of CTL that could accumulate in a peritoneal exudate. At the peak of the anti-tumour response, the largest number of CTL was found in the draining lymph node (l.66 × 105), followed by the spleen (3.47 × 104) and by a 24-hr casein-induced peritoneal exudate (6.01 × 103). Presumably, this greatly augmented production of CTL explains why C. parvum given intralesionally early enough during tumour growth can cause the regression of the weakly immunogenic P815 mastocytoma. [ABSTRACT FROM AUTHOR]

The mechanisms of non-specific unresponsiveness to contact sensitivity (CS) induced with intravenous (i.v.) administration of oxazolone (Ox)-conjugated syngeneic spleen cells was investigated. Non-specific suppressor cells were found in spleen cells of mice which had been injected i.v. with Ox-conjugated cells 7 days before. These non-specific suppressor cells blocked the efferent stage of CS profoundly, i.e. inhibited the activity of effector cells for picryl chloride (PCI). Since these suppressor cells were plastic-adherent and resistant to treatment with anti-Thy 1.2 antibody and complement, they were considered to be macrophage-like. These suppressor cells released non-specific suppressor factor(s) (NSF) during culture for 1 hr without any antigenic triggering. Effector cells for PCI which were treated with NSF for 1 hr at 4° lost their ability to transfer CS. NSF was easily absorbed by normal spleen cells. Furthermore, these NSF-treated spleen cells acquired the ability to inhibit the passive transfer of CS non-specifically. We also discussed the pathway for the induction of these macrophage-like suppressor cells. [ABSTRACT FROM AUTHOR]

Infection of nude mice with Mesocestoides cord results in spleen enlargement, with a decrease in percentage positive, but overall increase in Thy-1 positive cells. A low frequency of both parasite antigen-specific and alloantigen- reactive T-cell clones were isolated from the spleen, mesenteric lymph nodes, peritoneal exudate and Peyer's patches. The T-cell clones from spleen were found to express the antigens Thy- 1, Lyt-1 and Lyt-2; furthermore, after stimulation with antigen in the absence of exogenous T cell growth factor (IL-2), they produced normal levels of haemopoietic growth factor (IL-3), bone marrow proliferation activity and eosinophil differentiation activity. A higher proportion produced IL-2 compared to clones from normal mice. The isolation of antigen-specific 1-cell clones showing normal T-cell characteristics from young nude mice adds weight to suggestions that exposure to antigen results in significant extra-thymic T-cell maturation. [ABSTRACT FROM AUTHOR]

The suppressor T cell (Ts) function of old NZW, NZB, C57BL/6 and (NZB×NZW) F1 ((B/W)F1), mice to the 2.4-dinitro-1-fluorobenzene (DNFB) hapten was studied. Intravenous administration of dinitrophenyl (DNP) coupled syngeneic lymphoid cells (which normally induce DNP specific suppression) did not result in suppression of DNFB-specific contact hypersensitivity (CS) responses in old NZB or (B/W) F1 mice. Nevertheless, when spleen cells from these old mice were injected into young mice (either (B/W)F1 or A/Sn), strong suppression or the induction phase of CS responses was observed. In addition, effector phase suppressor activity was also observed when splenic cells from tolerized old (B/W) F1 donors were transferred into young (B/W) F1 mice during the effector phase of the CS response. In both cases, the significant ceils in the transfer were I-J+ T cells. Thus, the old mice retained functional Ts1 and Ts2 suppressor cells. However, the suppressive activity of the old mice could be reconstituted with spleen cells from primed young mice, suggesting that they have a defect in the Ts3 subset. This was further supported by the finding that the significant cells from the primed young mice were I-J positive and cyclophosphamide-sensitive. [ABSTRACT FROM AUTHOR]

Splenic B cells from older MRL/ Mp-lpr/lpr (MRL/1) and male BXSB mice responded to 2-mercaptoethanol (2-ME) in in vitro culture and generated immunoglobulin-secreting cells (IgSC). Optimal concentration of 2-ME to induce IgSC was 5 x 10-5 M. Kinetic studies revealed that the generation of IgSC was already apparent after 24 hr of culture and peak response was attained on the 2nd day. The response of B cells to 2-ME was enhanced in the presence of splenic T cells. Irradiation of B cells reduced the generation of IgSC. The B cell population of autoimmune mice responding to 2-ME to generate IgSC seems to be in a terminal stage of differentiation. This increased B cell differentiation was characteristic of autoimmune mice and assumed to have some significance in the development of autoimmune diseases. [ABSTRACT FROM AUTHOR]

Data presented in the study show that the cytotoxic factor (CF) produced in the spleen of (dengue-virus) infected mice can be purified by agarose-gel electrophoresis and Sephadex G- 100 gel filtration. CF is non-dialysable, heat-labile, trypsin- sensitive, unstable at acidic and alkaline pH, a macromolecular substance which sediments on ultra- centrifugation and is retained by a Millipore filter of 0.45 µm size. Its approximate molecular weight is 1.15 (± 0.34) × 105 as determined by gel filtration. [ABSTRACT FROM AUTHOR]

Specific pathogen-free B6D2 mice were infected intravenously with 108 viable BCG, M. habana or M. simiae and the level of tuberculin hypersensitivity to 2.5 μg PPD or cytoplasmic protein antigens (CPA) prepared from the other organisms was deter- mined using the footpad swelling test with increasing time after infection. This was correlated with the growth or persistence of mycobacterial populations within the liver. Spleen cells were removed from these infected mice and the level of blast transformation following exposure to PHA, PPD or M. habana or M. simiae CPA was measured in vitro. Early in the mycobacterial infections (day 14) thymidine incorporation by the spleen cells was significantly enhanced followed by a profound depression in incorporation rates as the infection progressed. The mechanism of this depressed response involved the production of suppressor T cells in the spleen. In the case of the M. simiae or M. habana infection, cells capable of mediating suppression were still present even after 12 months of infection. In the BCG infection, suppressor T cells declined with time so that by 4 months incorporation rates were back to normal and suppressor cells were no longer detectable in the spleens of the infected animals. [ABSTRACT FROM AUTHOR]

Cell-free extracts were prepared from the spleens of keyhole limpet haemocyanin (KLH)- primed, ovalbumin (OVA)-primed or unprimed mice, and were shown to suppress non-specifically the secondary antibody response to TNP1000KLH in vitro. Non-specific activity was retained on storage in liquid nitrogen and was not removed by dialysis. These findings are in contrast to those reported elsewhere where it was found that cell-free extracts from the spleens of KLH-primed mice specifically suppressed the IgG response to TNP1000KLH; this discrepancy may well lie in the initial immunological status of the mice used. Nevertheless it is concluded that proper account should be taken of non-specific suppressive effects when studying the activity of cell-free extracts in vitro. [ABSTRACT FROM AUTHOR]

Mouse spleen lymphocytes were stained with rabbit antisera specific for either μ chain or δ chain, followed by fluorescein-conjugated goat anti-rabbit immunoglobulin. The cells were analysed and fractionated using a fluorescence-activated cell sorter. Fifty-five per cent of the lymphocytes stained with a polyspecific anti-Ig reagent or with a combination of anti-μ and anti-μ reagents, while about 40% of the lymphocytes were stained when either the anti-δ reagent or the anti-δ reagent was used alone. Three per cent of the lymphocytes stained with the anti-δ reagent, but not with the anti-δ reagent, and eight per cent stained only with the anti-δ reagent. Unfractionated spleen cells and populations depleted of p-or δ-bearing cells were cultured in the presence of lipopolysaccharide. All three populations responded by incorporating [³H]-thymidine and secreting IgM and IgG. Spleen cells were fractionated by a rosetting technique into complement receptorpositive and negative populations. Both populations were able to respond to lipopolysaccharide and to synthesize Ig of both the IgM and IgG classes. Unfractionated cells and complement receptor-negative populations were stained for surface μ or δ chain and analysed on the fluorescence-activated cell sorter. The distribution of staining intensity suggested that the complement receptor-bearing population was enriched in cells which stain weakly for μ and cells which stain with a low to intermediate intensity for δ chain. It is concluded that the precursors of IgM- and IgG-secreting cells are not limited to any one of the three populations of cells defined on the basis of surface immunoglobulin or to either of the populations defined on the basis of the complement receptor. [ABSTRACT FROM AUTHOR]

In assay cultures of normal mouse spleen cells stimulated with sheep erythrocytes (SRBC) on day 0, the addition on day 2 of murine enhancing factor (MEF), purified from supernatants of mixtures of allogeneic lymphoid cells, resulted in significant non-specific augmentation of the haemolytic plaque response. A different supernatant activity, antibody initiation suppressor factor (AISF), generated by specific anamnestic stimulation of spleen cells from mice immunized 7 days earlier with horse erythrocytes (HRBC), was capable of significant inhibition of the anti-SRBC plaque-forming cell response of similar assay cultures when added simultaneously with SRBC on day 0 of a 5-day culture period. The addition of optimal concentrations of both MEF and AISF to SRBC-stimulated spleen cells at the initiation of culture blocked the inhibitory activity of the suppressive mediator. Similarly, the activity of MEF in enhancing both the AISF-specific (anti-HRBC) and non-specific (anti-SRBC) responses was abrogated in the presence of AISF added in optimal amounts with MEF on day 2 of culture. A similar antagonistic interaction between AISF and MEF was observed when each factor was added, at its respective optimal dilution and time, to SRBC-stimulated splenocytes in vitro. More- over, AISF, a factor derived from antigen-activated T lymphocytes, failed to replace suppressor T-cell function in assay cultures of T-cell depleted mouse splenic B cells. Paradoxically, AISF markedly enhanced the anti-SRBC haemolytic plaque response of such cultures. The addition of graded numbers of T lymphocytes to AISF-treated B-cell cultures also resulted in an augmented humoral immune response. But the target cell of AISF was the macrophage, since the presence of splenic macrophages in the B-cell cultures reconstituted the suppressive activity of the inhibitor. In addition, AISF dramatically increased the number of macrophage-like colony-forming units in bone marrow cultures in vitro. These data are discussed in terms of the delicate balance and possible interactions between effector molecules which, in part, regulate antibody synthesis. [ABSTRACT FROM AUTHOR]

Maturation of neonatal spleen cells was studied in vitro with a cell population restricted with respect to functional properties. It is shown that the onset of the immune response to SRBc in post-natal mice was delayed because B and T cells were incompetent. It appears, however, that the development of these two cell populations does not occur in parallel. Since the addition of adult macrophages failed to overcome the incompetence of neonatal B cells in the presence of a T cell replacing factor, it is suggested that the late appearance of immune competence is due to the inability of B cells to process a T-cell signal. [ABSTRACT FROM AUTHOR]

A single intraperitoneal injection of 0.5 mg methotrexate (MTX) has been found to increase the immune reactivity of spleen fells from (C57B1/6 ⊗ DBA/2)F1 mice. Five days after injection, spleen fells from MTX-treated mice exhibited greater PHA responsiveness and GvH reactivity, and mice given SRBC at this time developed greater than normal direct PFC responses. This pattern of effects of MTX was particularly evident in mice that had been given high doses of BCG intravenously 14 days before testing, a treatment that generally depressed the measured activities. MTX enhancement of GvH was also observed in mice that had been depleted of short-lived T lymphocytes by adult thymectomy. We suggest that MTX-sensitive cells possibly exert, particularly in BCG-treated mice, a suppressive action on the responding cells. [ABSTRACT FROM AUTHOR]

In the spleen of mice immunized with bromelin treated rat red blood cells (R.BC), the number of PFC against bromelin treated isologous RBC increased, although immunization with non-treated rat R.BC or bromelin treated isologous RBC gave no increase in number of these PFC. This immune response was found to be T-independent and the PFC developed are exclusively of direct or Ig-M type. In the secondary immune response, production of these PFC was depressed rather than increased. This can be thought of as one of the defence mechanisms against overproduction of autoantibodies in confrontation to foreign antigens cross-reactive with self antigenic determinants. This depressed secondary immune response can be adoptively transferred by primed spleen cells but no active suppressor effect was found. We concluded that clonal elimination by exhaustive differentiation may be operative in this depressed secondary immune response. [ABSTRACT FROM AUTHOR]

Splenic immune responses having varying dependence on accessory cell co-operation have been studied after infection of mice with Friend virus. Infection has no effect on cell proliferation or antibody production in cultures stimulated with E. coil lipopolysaccharide. The response in vivo to type III pneumococcal polysaccharide is depressed only moderately. The response to sheep red blood cells is depressed severely both in vivo and in vitro. Depression in vitro is greatly reduced by co-stimulation with E. coil lipopolysaccharide. Depletion of potential suppressor lymphocyte populations by irradiation or adult thymectomy does not ameliorate depression of responses to sheep red blood cells or pneumococcal polysaccharide. Responses after adult thymectomy plus irradiation are not affected by the virus. Although it is known that macrophage and helper T-lymphocyte co-operation are not themselves impaired by infection, these results suggest that there is a direct relationship between severity of immune expression and dependence on co-operation. Implications for the action of the virus are discussed. [ABSTRACT FROM AUTHOR]

Determines the conditions in which unprimed or resting memory cells might be stimulated in vitro to produce antibodies of different classes. Comparison between Mishell-Dutton and modified Marbrook culture systems in terms of their ability to support an immune response by quiescent memory cells exposed to antigen in vitro; Development of immunoglobulin M and G-antibody secreting cells from primed mouse splenocytes in vitro; Implications on cellular differentiation of immune response studies.

Male and female spleen cells were compared before and after culture for their cytotoxicity to autologous-embryo fibroblasts. Cultures of male cells developed significantly greater reactivity than cultures of female cells. Moreover, while the cytotoxicity derived from cultures of male cells was totally abolished by treatment of effector cells with a mouse anti-T cells antiserum, such an antiserum had less affect on the effector cells of female mouse cultures. [ABSTRACT FROM AUTHOR]

We have investigated the ability of male-mouse spleen cells before and after culture in the absence of deliberate antigenic stimulation to show specific cytotoxicity to syngeneic embryo-fibroblast cells. The data suggest that cytotoxicity which develops spontaneously in such spleen cell cultures is directed primarily against embryo-associated antigens. Syngeneic Con-A-stimulated spleen cells, which, unlike fresh normal spleen cells, are also lysed by rabbit anti-mouse embryo antisera, are also a suitable target to demonstrate spontaneously developing cytotoxicity. [ABSTRACT FROM AUTHOR]

Spleen cells from normal BALB/c mice showed in vitro proliferative response against hapten-conjugated syngeneic spleen cells. Trinitrophenylated (TNP) spleen cells were prepared by treating normal spleen ceils with sodium 2,4,6-trinitrobenzenesulphonate (TNBS). Four-day cultures of TNP-labelled spleen ceils incorporated 2·5–7·4 times more [3H]thymidine than similar cultures of un- treated spleen cells. An obviously positive mixed lymphocyte reaction (MLR) by normal spleen cells against mitomycin C(MC) treated TNP-labelled syngeneic spleen cells was observed after 4 days of culture. The MLR to TNP-labelled syngeneic cells was inhibited in the presence of ε-TNP-L-lysine by 23–37%. The spleen cells from the mice injected intraperitoneally with TNP-labelled syngeneic spleen cells showed a higher MLR against TNP-labelled spleen cells than normal spleen cells. The sensitized spleen cells also showed an increased response to MC-treated spleen cells. These results suggest that normal spleen cells include cells which can recognize the hapten and new antigenic determinants introduced into syngeneic spleen cells by chemical modification. [ABSTRACT FROM AUTHOR]

Analysis of the electrophoretic mobility of mouse thymus cells has showed two main populations, with mean mobility values of 0·77±0·023 μm s-1 V-1cm and 0·99±0·015 μm s-1V-1cm; these absolute values varied slightly from one strain to another. Implantation of tumour cells caused the relative proportions of these two populations to change dramatically within 48 hours, when an increase in the fast-moving ‘immunocompetent’ thymocytes was observed. The ratio of slow to fast cells changed from 9:1 in the normal BALB/c animal to 2:1 in the presence of the tumour cells and this 2:1 ratio persisted throughout the remainder of the animal's life. However, inoculation of histocompatible spleen cells from a normal individual evoked only a brief response in the host's thymus. This change in ratio of slow to fast cells in the thymus was interpreted as an increased production of immunocompetent cells in response to the presence of the tumour cells. [ABSTRACT FROM AUTHOR]

The recovery of the B-cell population in adult thymectomized, BM mice) was studied. The irradiated and bone marrow-reconstituted mice T x number orb cells in the spleen of T × BM mice was estimated at various times after bone marrow transplantation. The spleen cells to be tested were mixed with dexamethasone-resistant thymocytes (DRT) and sheep red blood cells (SRBC) and transferred to irradiated recipients. The number of plaque-forming cells (PFC) in the spleen of the recipients was determined 7 days later. Using this functional B-cell assay a sequential appearance of the precursors of IgM-, IgGand IgA-PFC in the spleen of T × BM mice was observed. The precursors of IgM-PFC (IgM-B cells) were present immediately after transplantation. The first IgG-B cells could be detected at 13-16 days after transplantation and the IgAB cells finally appeared at 22 days after transplantation. The number of B cells reached a constant and normal level at 30 days after transplantation. The IgM-. IgG- and IgA-B cell development in sham-thymectomized, irradiated and bone marrow-reconstituted mice (ST × BM mice) was virtually the same as in T × BM mice. [ABSTRACT FROM AUTHOR]

A migration inhibition test on spleen explants from NZB mice of different ages (4, 8 and 12 months) was performed using 40 μg/mI of DNA. The results have shown increased positivity with ageing of experimental animals. [ABSTRACT FROM AUTHOR]

Spleen colonies arising in irradiated mice pre-immunized with sheep and chicken erythrocytes were assayed for the content of 19S-and 7S-producing cells. This was done by applying to each isolated colony a direct (19S) and an indirect (7S) plaque test for each antigen. In both tests, the positive anti-sheep or anti-chicken reactivity of cells was found to be completely independent of each other. The plaque forming capacity for 19S- and 7S-producing cells showed a marginally significant association in the anti- sheep response only. The numbers of plaque forming cells were distributed exponentially, suggesting that 19S- and 7S-producing cells have similar growth kinetics. These results confirm the clonal nature of antibody response within spleen colonies, thus making the system amenable for genetic studies of antibody production. They document a strictly vertical transmission of antibody specificity, and possibly of immunoglobulin class. The incomplete independence of 19S and 7S distribution is interpreted as due to local or technical conditions rather than to a production ‘shift’ of the clones. [ABSTRACT FROM AUTHOR]

Treatment of mice with hydrocortisone reduced the number of thymus-cells to 6 per cent and the number of spleen-cells to 32 per cent of matched controls. Per spleen, an absolute decrease of cells carrying immunoglobulin receptors on the surface as determined by immunofluorescence was found and a smaller decrease in cells with theta-antigen. Equal numbers of thymus and spleen cells from hydrocortisone-treated mice and matched controls were cultured and stimulated with phytohaemagglutinin (PHA), pokeweed mitogen (PWM), allogeneic cells, and, following immunization, with keyhole limpet haemocyanin (KLH). Stimulation was assessed by incorporation of [³H]thymidine into the acid precipitable fraction of the cultured cells. With thymus cells, hydrocortisone treatment increased the reaction to PHA and allogeneic cells. Thymus cells from untreated animals already gave a good response to PWM, further increased by treatment with hydrocortisone. With spleen cells, hydrocortisone treatment reduced the reaction to KLH, PWM, allogeneic cells and PHA in decreasing order. The results are discussed with reference to cells affected by hydrocortisone treatment and to the mechanism of in vitro lymphocyte stimulation. [ABSTRACT FROM AUTHOR]

Male CBA mice were given a single intraperitoneal injection of sheep red blood cells (SRBC) or horse red blood cells (HRBC). They were killed at intervals of 1–10 days thereafter, and micro-cultures of spleen cells or peritoneal cells (PC) were prepared. These consisted of a thin film of tissue culture medium containing carboxymethyl cellulose (CMC), mouse lymphoid cells, guinea-pig complement and either SRBC or HRBC, held at 37° under liquid paraffin. Cultures were read repeatedly for appearance of haemolytic plaques. PC from SRBC-immunized mice showed an altered reactivity on SRBC monolayer cultures. The peak plaque count achieved in vitro fell progressively for 4 days after immunization, and then returned to normal by day 7. The actinomycin D resistant component of the PC response rose rapidly; at 1 day after immunization it was equal to the total response. Over the next 3 days after immunization it fell again to normal levels. The results suggested that the in vivo injection sets in train events locally in the peritoneal cavity which resembled those following in vitro culture of normal PC in SRBC monolayers. The effects were immunologically specific as only marginal changes followed the injection of HRBC. Spleen cells from SRBC-immunized mice, when cultured in SRBC monolayers, yielded many cells capable of giving plaques after 5–60 minutes incubation, as expected. These were deemed to be cells forming antibody at the moment of killing of the animal. In addition, such cultures developed new plaques over the subsequent 23 hours in culture. These were produced by cells not initially forming antibody which switched into antibody secretion at some time during culture. At early time points after immunization, this second type of cell was much more numerous than the first type. The switch from non-secretor status could occur in the presence of a high concentration of actinomycin D. Operationally these nonsecretors in immunized spleens resembled an important fraction of PC from unimmunized retired breeder mice. The progressive conversion of non-secretor cells into secretors, if it occurs in vivo, would have a major influence on the kinetics of appearance of PFC in a spleen after immunization. While spleen cells from mice immunized with HRBC performed on HRBC monolayers much as described above, PC from HRBC-immunized mice could not be induced to cause significant lysis in HRBC monolayers. The same was true of PC from mice chronically fed with HRBC. In fact, no method has yet been found to persuade PC to produce lytic plaques active against erythrocytes other than SRBC. [ABSTRACT FROM AUTHOR]

Proteolytic events might play a role during lymphocyte activation. Mouse spleen cells were therefore stimulated in serum-free cultures by PHA, ConA, LPS and dextran sulphate and the effect of various added protease inhibitors on [³H]-thymidine incorporation investigated. Both soybean inhibitor and Trasylol inhibited the response of the cells to all mitogens. The other inhibitors (antipain, leupeptin, ovomucoid, alpha-1 trypsin, alpha-2 macroglobulin) had little or no effect. The marked inhibitory effect of tosyl-lysine chloromethylketone could be neutralized by reduced glutathione, indicating an effect on intracellular glutathione rather than on proteases. [ABSTRACT FROM AUTHOR]