Maturation of the lymphoid system IV. Ontogenetic compartmentalization of T cell function

Pretreatment of one day old but not eight day old or adult A/J mice with soluble ovalbumin (OVA) initiated specific T-cell unresponsiveness as reflected both in T-cell dependent cellular proliferation and in anti-hapten antibody responses to dinitrophenyl-OVA. In contrast, injection of soluble human gamma globulin into either neonatal or adult A/J mice resulted in unresponsiveness.The ability of lymph node T-cells to be sensitized by protein antigens occurred shortly after birth, since the degree of sensitization in 9 and 26 day old mice was similar. Finally, a striking ontogenetic difference was noted in the capacity of lymphocytes derived from the lymph nodes and spleens of young mice to respond to T-cell mitogens. Thus, while splenocytes obtained from 9 day old mice exhibited meager responses to PHA and Con A, lymph node cells from these animals responded at nearly adult levels. These observations are interpreted as reflecting an ontogenetic and tissue-specific division of T-cell function.     

Adoptive transfer of resistance to Nematospiroides dubius infections of mice and an assay to measure the in vitro proliferation of lymphocytes reactive with N. dubius antigen.

Spleen and mesenteric lymph node cells from Nematospiroides dubius-infected and normal control mice were cultured in vitro with N. dubius antigen. Proliferation of these cells in response to antigen was measured by the uptake of [3H]TdR. Cells harvested from mice during a primary infection did not proliferate in vitro; however, low levels of specific proliferation could be demonstrated if these mice were treated on Day 5 post-infection with 20 mg/kg of cyclophosphamide i.p. A strong cell proliferative response was measured 6 days following a challenge infection; spleen cells responded more strongly than cells from the mesenteric lymph nodes (MLN), but the addition of lymph node cells to spleen cell cultures did not suppress the latter response. Responsiveness of spleen cells to concanavalin A (Con A) was two-fold higher in infected mice than in normal controls, but the proliferation of MLN cells to Con A was similar in infected and uninfected mice. When N. dubius-resistant B10.M (H-2f) mice were compared to the susceptible B10.BR (H-2k) mice, no differences were observed in the spleen cell response to N. dubius adult antigen following challenge infections. However, after a tertiary infection, MLN cells from the resistant strain proliferated strongly in comparison to cells from susceptible mice. Spleen or MLN cells from resistant mice transferred immunity to naive recipients provided that the recipients had received a prior injection i.p. with adult N. dubius antigen. The injection alone, or cells in the absence of the injection, failed to protect the recipients from N. dubius challenge.     

Differential effects of marijuana components on proliferation of spleen, lymph node and thymus cells in vitro

The effects of delta 9 THC and 11-OH THC on the proliferative response of murine spleen cells stimulated in vitro with the T cell mitogens Con A or PHA were compared with the effects of these drugs on the mitogen-induced proliferation of murine thymus and lymph node cells. Thymus cells were found to be suppressed at lower cannabinoid concentration than either spleen or lymph node cells. However, splenic cells were more easily suppressed than were the lymph node cells. Lymphoid cell numbers were varied from 1 X 10(6) to 8 X 10(6) cells and treated with a constant dose of either THC or 11-OH THC. When suppression was noted with spleen and lymph node cells, the smallest number of cells in the assay resulted in the greatest level of suppression of cell proliferation. No significant suppression to PHA induced proliferation was found for lymph node cells at any cell number tested. Thymus cells were always more readily suppressed than spleen or lymph node cells regardless of the number of cells in culture. Furthermore, 11-OH THC suppressed the responsiveness of the thymus cells to PHA more than to Con A under the experimental conditions used. Thus, the ability of cannabinoids to induce suppression of the proliferative response of lymphoid cells to mitogens depends on the organ source of the cells, nature of the cannabinoid (THC or 11-OH THC), dose of the cannabinoid, mitogen used (PHA or Con A), and number of cells in culture.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunological Properties of Peripheral Rat Lymphocyte Subpopulations Reacting Selectively with Guinea Pig Complement

Functional properties of peripheral rat T lymphocytes selectively affected by reaction with guinea pig serum (GPS) complement (C) were studied in this work. Cells sensitive to GPS cytotoxicity represented 2-7% of the nucleated cells in the spleen and 1-4% in lymph nodes. Responses of spleen and lymph node cells to Con A and PHA were markedly reduced following treatment with GPS whereas LPS-induced responses were not altered. GPS treatment also abrogated both the specific response of lymph node cells to a protein antigen and the production of leukocyte migration inhibitory factor by splenic cells. By contrast, GPS treatment significantly increased the reactivity of spleen and lymph node cells to alloantigens and enhanced the in vitro immune responsiveness of splenocytes to sheep erythrocytes. These results highlight the selective reactivity of guinea pig C with discrete subsets of immunoregulatory peripheral rat T lymphocytes.     

Lymphocyte-mediated cytotoxicity against tumor cells. III Differentiation of concanavalin A-activated cytotoxic effector cells.

The maturation of selected T cell responses in the lymphoid organs of irradiated CBA mice was followed after adoptive transfer of syngeneic fetal liver cells. Mitogenic responsiveness to phytohemagglutinin (PHA) and concanavalin A (Con A) was found to reach control values 3 weeks after reconstitution in the thymus, spleen, and lymph node, of fetal liver repopulated animals. Spleen and lymph node cell reactivity in mixed lymphocyte culture reactions required 6 weeks to reach significant values. However, the ability of spleen cell suspensions to be activated by Con A into cytotoxic effector lymphocytes appeared after only 2 to 3 weeks. It is concluded that two functionally distinct T cell subpopulations exist in the spleen, one which can be activated into cytotoxic effector lymphocytes by Con A, and one which responds to alloantigens by DNA synthesis.     

Immunological Properties of Peripheral Rat Lymphocyte Subpopulations Reacting Selectively with Guinea Pig Complement

Functional properties of peripheral rat T lymphocytes selectively affected by reaction with guinea pig serum (GPS) complement (C) were studied in this work. Cells sensitive to GPS cytotoxicity represented 2-7% of the nucleated cells in the spleen and 1-4% in lymph nodes. Responses of spleen and lymph node cells to Con A and PHA were markedly reduced following treatment with GPS whereas LPS-induced responses were not altered. GPS treatment also abrogated both the specific response of lymph node cells to a protein antigen and the production of leukocyte migration inhibitory factor by splenic cells. By contrast, GPS treatment significantly increased the reactivity of spleen and lymph node cells to alloantigens and enhanced the in vitro immune responsiveness of splenocytes to sheep erythrocytes. These results highlight the selective reactivity of guinea pig C with discrete subsets of immunoregulatory peripheral rat T lymphocytes.     

Immunosuppression and cytokine production in mice infested with Ixodes ricinus ticks: a possible role of laminin and interleukin-10 on the in vitro responsiveness of lymphocytes to mitogens.

T cells from BALB/c mice infested 9 days before with Ixodes ricinus nymphs had a suppressed response to in vitro concanavalin A (Con A) stimulation compared to cells from uninfested mice. When laminin (the main component of the extracellular matrix) was used as a coating agent, the Con A response of naive mice was characterized by a decrease in cell proliferation, whereas there was no significant effect on the mitogen response of cells from infested mice. In contrast, an increased response to lipopolysaccharide (LPS) was observed when assaying lymph node cells of infested mice, probably reflecting an increase in B-lymphocyte number or activity. LPS cell stimulation was not modified by laminin. Supernatants of lymph node cells, taken 9 days after the first infestation of mice, stimulated with Con A in vitro, contained interleukin-10 (IL-10) but no significant levels of IL-5 as tested by enzyme-linked immunosorbent assay. At this stage of the infestation all T cells reactive with tick antigens generated in lymph nodes that drain the tick fixation site, were CD4+ cells, as determined by CD4+ depletion. With cells taken 9 days after the third infestation an increase of IL-5 and IL-10 was observed. The IL-10 levels were higher than the IL-5. According to these observations, we conclude that the reduction of T-cell proliferation in response to Con A observed in lymph node cells from infested mice, may be due to the combined effect of laminin interaction with T lymphocytes during migration and IL-10 production by these lymphocytes.     

Positive selection of T-cell subsets. I. Proliferative responses of Lyt 2 separated thymocytes and splenic T cells.

Flow microfluorometry analysis of peanut lectin non-agglutinable (PNA-) thymocytes (ThC) reveals the existence of 30%-50% Lyt 1,2,3+ and 50%-70% Lyt 1+,2,3- subpopulations. Using positive selection on anti-immunoglobulin-coated (Mage) plates, we selected PNA- Lyt 2+ and PNA- Lyt 2- ThC as well as their peripheral counterparts in the spleen. These populations were tested in parallel for their ability to respond to concanavalin A (Con A) and phytohaemagglutinin (PHA), to respond to allogeneic stimulation in the mixed lymphocyte reaction (MLR); ThC subpopulations were also tested for their ability to provide synergy with lymph node cells (LNC) in the MLR. It was found that (a) Lyt 2- cells of both thymic and splenic origin responded to all doses of Con A or PHA; (b) PNA- Lyt 2+ ThC were unresponsive to Con A or PHA, whereas splenic Lyt 2+ T cells responded to low doses of mitogens; and (c) PNA- ThC of both Lyt phenotypes responded in a MLR and provided synergy with LNC in the MLR. These data support the notion that Lyt 2+ cells of either PNA- or PNA+ subpopulations must undergo post-thymic maturation before becoming responsive to low doses of T-cell mitogens.     

Age-related changes of mitogen responsiveness in different lymphoid organs from outbred NMRI mice

Lymphocytes from spleen, thymus and lymph nodes from individual young adult (3--4 months) and aged (26--30 months) NMRI mice were stimulated with the mitogens Con A, PHA and LPS. 24 hours later, the number of cell with increased RNA-content (G1 cells) was determined by cytofluorometry. In parallel the 3H-thymidine incorporation after 48 hours was measured for the same cell samples. Aged animals in average produced less G1 cells and incorporated less 3H-thymidine as compared to young adults. By calculating the 3H-thymidine incorporation per G1 cell, the proliferative capacity of mitogen-induced G1 cells can be estimated. These ratios are lower in aged mice as compared to young adult, suggesting that in these animals not only less cells can be activated as measured by cytofluorometry, but also from these activated cells again fewer continue the cell cycle by initiating DNA-synthesis. In response to Con A and PHA, aged mice in average produce less G1 cells in all of the three lymphoid organs tested. In response to LPS, however, the young adult produced only few G1 cells in lymph nodes and practically none in thymus, whereas in aged animals a considerable number of G1 cells was found in both organs. Corresponding results were found for the 3H-thymidine incorporation. These results indicate that in addition to the reduction of the mitogen-response an age-related change in the distribution of mitogen-responsive cells in the different lymphoid organs takes place.     

Immunological Marker Analysis of Mitogen-Induced Proliferating Lymphocytes Using Brd U Incorporation or Screening of Metaphases

The proliferative effects of the mitogens phytohaemagglutinin (PHA), concanavalin A (Con A), pokeweed mitogen (PWM), and staphylococcal protein A (SpA) were investigated using two different methods which enable immunological marker analysis of proliferating cells: either surface marker labelling followed by BrdU incorporation or screening of metaphases after surface marker labelling. Therefore peripheral blood mononuclear cells from six healthy volunteers were stimulated with these four mitogens. Both PHA and Con A gave rise to more CD8+ than CD4+ proliferating cells. PHA, but not Con A, induced B-cell proliferation as well. PWM mainly caused T-cell proliferation. SpA also appeared to be a potent T-cell mitogen in addition to its capacity to induce B-cell proliferation. However, in contrast to the other mitogens SpA predominantly stimulated CD4+ cells.     

Features of renal vasculitis in autoimmune MRL lpr/lpr mice: phenotypes and functional properties of infiltrating cells.

MRL lpr/lpr (MRL/1) mice spontaneously develop a widespread renal vasculitis. The majority of the cells in vasculitic lesions are bright Ly-1, L3T4 and la-positive in contrast to the cells found in lymph nodes and spleens of the old MRL/1 mice. However, despite differences in phenotypical patterns, B and T cells from arteritic lesions do not differ from mononuclear cells (MNC) eluted from MRL/1 lymph nodes with regard to the frequency of IgG secreting cells and the proliferative responses to Concanavalin A (Con A). Co-culture experiments with congeneic MRL+/+ (MRL/n) spleen cells indicate that the poor response to Con A of the MNC eluted from vasculitic lesions is, unlike the case of lymph node MNC, due to suppressive action of vasculitic cells on the indicator cell population. Further support for the activation status of infiltrating MHC in kidney vasculitic lesions, expressed by high in vivo uptake of 3H-thymidine, was obtained by autoradiography performed on frozen sections. The observed differences in phenotypic patterns and functional features between lymph node MNC and infiltrating vasculitic MNC indicate that different immune mechanisms may be responsible for the development of lymphadenopathy and vasculopathy, respectively in MRL/1 mouse.     

Treatment of autoimmune MRL/lpr mice with anti-B220 monoclonal antibody reduces the level of anti-DNA antibodies and lymphadenopathies.

The predominant accumulating cells in the lymphoid tissue of MRL/lpr mice have been shown to carry the B220 cell marker. This antigen is expressed on B cells and on T-cell precursors. In order to know the pathogenic involvement of cells carrying this marker, we treated MRL/lpr mice periodically with RA3-6B2, a specific anti-B220 monoclonal antibody, or rat IgG2b as a control. After 12 weeks of treatment, a significant reduction (P less than 0.01) in the size of the lymph nodes and spleen was observed only in the group treated with RA3-6B2 monoclonal antibody. This reduction was mainly due to a decrease in the Thy-1.2+ and B220+ subpopulations. Anti-DNA and anti-Sm antibody titres were also reduced significantly (P less than 0.01) after the therapy. Proliferative response to mitogens (Con A, PHA, LPS) and IL-2 production was not improved after the anti-B220 treatment. These results suggest a pathogenic role of lymphocytes carrying the B220 marker in the autoimmune disease of MRL/lpr mice.     

Lymphatic mast cells in response to in vitro stimulation by non-specific T-cell mitogens.

Activation of lymph nodes by the T-cell mitogens PHA and Con A is correlated with a depletion of lymphatic mast cells. This result, and our earlier reports on the depletion of mast cells in lymph nodes stimulated by allogeneic and tumour cells, as well as on the elevation of mast cell number in thymus-less 'nude' mice, lead us to the conclusion that antigen- or mitogen-stimulated T lymphocytes produce lymphokine(s) which degranulates mast cells and/or is responsible for their negative chemotaxis.     

Increased leukocyte diversity and responsiveness to B-cell and T-cell mitogens in cell suspensions prepared by enzymatically dissociating murine lymph nodes

The isolation of viable follicular dendritic cells (FDCs) from murine lymph nodes requires that the nodes be enzymatically dissociated with collagenase and the protease dispase. The present study was undertaken to compare leukocyte populations derived from enzymatically dissociated and mechanically disrupted lymph nodes. We examined cell viability, cell number, cell types, and the proliferative response to the mitogens LPS, PHA, and Con A. Cells were prepared by taking the inguinal, brachial, axillary, and popliteal lymph nodes from one side of the animals and mechanically disrupting them. The corresponding lymph nodes from the other side of the same animals were digested with enzymes. The enzyme-treated lymph nodes yielded substantially more cells and had a higher percentage of viable cells. Over 40% more viable cells were available for cell culture using the enzyme dissociation method. The numbers of FDCs, macrophages, plasma cells, and fibroblasts were clearly increased. The cells dispersed by enzymatic means responded better than the mechanically dispersed cells to both B-cell and T-cell mitogens. This was especially striking in cultures at lower cell densities. We conclude that the method of cell dissociation has a marked effect on the types of viable cells released and available for culture, as well as on the ability of the cells to respond. We believe the cell types released by enzymatic dissociation and their response in culture more accurately reflect conditions in vivo.     

Murine immune response to HIV-1 p24 core protein following subcutaneous, intraperitoneal and intravenous immunization.

The murine immune response to baculovirus-produced human immunodeficiency virus type-1 (HIV-1)p24 was examined after injection by three different routes: subcutaneously (s.c.), intraperitoneally (i.p.) and intravenously (i.v.). Both antigen-specific T-cell proliferation and serum antibody were induced by i.p. injection. In contrast, s.c. and i.v. injection of antigen resulted in specific antibody generation alone. Lympho-proliferative responses seen after i.p. injection were confined to splenocytes, and were greater after a low dose of antigen than after a high dose. p24-specific proliferation was not detected in lymph node cells. CD4:CD8 ratios were normal in lymph nodes and spleen at all times, irrespective of the dose or route of administration. p24-specific serum IgG antibodies were detected in all animals after the second injection of antigen. After s.c. and i.v. administration of high doses of antigen, the median antibody titres continued to rise after a third injection, but plateaued in animals injected by the i.p. route. In contrast, low doses of antigen given i.p. increased the median titre during and after the course of four injections. A low antigen dose given s.c. resulted in a plateau of median titre between the third and fourth injections. In i.v.-injected animals the median titre decreased between the third and fourth injections. IgG1 p24-specific antibodies were detected in all immunized mice, whereas IgM antibodies were detectable only following i.p. injection of antigen.     

Resistance to Mycobacterium lepraemurium is correlated with the capacity to generate macrophage activating factor(s) in response to mycobacterial antigens in vitro.

The kinetics of cell-mediated immunity developed during the course of Mycobacterium lepraemurium infection were determined in resistant (C57BL) and susceptible (BALB/c) mice. Control of M. lepraemurium growth following footpad infection was T-cell dependent in C57BL mice as shown by the finding that T-cell deprived mice had enhanced bacterial counts in the footpad. In contrast, T-cell deprivation did not significantly alter the course of infection in BALB/c mice. However a T-cell dependent inflammatory response, resulting in an increase in size of the infected footpad, occurred in both strains, although it developed slightly later in BALB/c mice. Cells isolated from the lymph nodes, draining the infected foot-pads, were assayed for their proliferative responses to heat-killed M. lepraemurium (HK-MLM) antigens. Although lymph node cells from both mouse strains proliferated to HK-MLM early in the infection (1-2 weeks) both C57BL and BALB/c mice developed diminished in vitro proliferative reactivity within 4-6 weeks post-infection. Supernatants derived from cultures of lymph-node cells that had been stimulated with concanavalin A (Con A) or HK-MLM antigens, were assayed for the presence of macrophage-activating factor (MAF) activity using a tumour cytostasis assay and interferon (IFN) activity using a viral growth inhibition assay. Significantly higher levels of MAF and IFN were found in culture supernatants deprived from HK-MLM stimulated lymph-node cells from infected C57BL mice than from BALB/c mice during the first 8 weeks of infection. However, cells from infected mice of both strains produced similar amounts of both MAF and IFN in response to Con A.     

In vivo effects of an acute nonimmunological inflammation in rats on the lymphoproliferative response to mitogens

Lymphoid cells (spleen, lymph node and thymus) derived from rats after induction of an acute nonimmunological inflammatory reaction responded to various mitogens (Phytohemagglutinin, PHA; Concanavalin A, Con A; Lipopolysaccharide, LPS) with increased proliferation when compared with cells derived from normal animals. In the absence of mitogens, lymphoid cells from animals undergoing an acute nonimmunological inflammation demonstrated enhanced proliferation compared with cells from normal animals. These results clearly demonstrated that during acute nonimmunological inflammation the reactivity of lymphoid cells was increased.     

Maturation of mitogenic response in foetal guinea-pig.

Lymphocytes obtained from thymus, spleen and heart blood of 33-day-old guinea-pig foetuses and from lymph nodes of 48-day-old foetuses onwards were stimulated by phytohaemagglutinin (PHA). concanavalin A (Con A) and dextran sulphate (DxS). The results, based on the analysis of ninety-five foetuses, indicated that the mitogenic responses of the guinea-pig T cells matured first in the thymus and then in the spleen and lymph nodes, in that order. The PHA and Con A responses in the thymus, spleen and lymph nodes and the DxS response in the spleen improved with the increasing age of the foetus. A clear improvement of the mitogenic responses did not take place in the blood. Thus, at birth the guinea-pig is immunologically mature as regards the PHA and Con A responses in the thymus, spleen and lymph nodes and the DxS response in the spleen. Regarding the maturation of PHA and Con A responses by lymphocytes from the blood, further studies are needed, since at birth both of these responses are clearly below the adult level.     

Lymphocyte function in experimental endemic syphilis of Syrian hamsters.

We have studied the changes in the lymph nodes, spleen and thymus that occur in inbred LSH Syrian hamsters infected with Treponema pallidum Bosnia A, the causative agent of endemic syphilis, as well as the B-cell responses of these infected animals to helper T-cell independent and dependent antigens. The lymph nodes increased significantly in weight up to 6 weeks after infection, and contained viable treponemes. No significant changes in the spleen weight were observed, and no viable treponemes could be recovered from the spleen. However, the size of the thymus decreased steadily during the course of the disease. The relative number of Ig+ cells (B cells) increased in the spleen and regional lymph nodes, whereas the relative number of T cells decreased during the course of infection. In both the spleen and lymph nodes, the relative number of macrophages increased initially and decreased thereafter in the form of a bell-shaped curve showing a peak at 4-6 weeks of infection. The ability of splenic lymphocytes from infected hamsters to mount a primary PFC response to pneumococcal polysaccharide type III (SIII), a helper T-cell independent antigen, was elevated throughout the course of infection. However, the splenic PFC response to sheep erythrocytes (SRBC), a helper T-cell dependent antigen, was increased only during the first 4 weeks of infection and progressively decreased thereafter. The PFC responses of infected lymph node lymphocytes to both SIII and SRBC were increased during the first 4 weeks and decreased thereafter. These data suggested that atrophy of the thymus seen in syphilitic infection is accompanied by the complex losses of subsets of T cells and altered B-cell functions. An early loss of suppressor T cells in both the lymph nodes and spleen occurs concomitantly with a loss of T helper cells and heterologous (treponema-unrelated) B-cell functions in the lymph nodes. Helper T cells are lost from the spleen only in the later stages of infection, whereas splenic B-cell functions remain intact throughout the course of the disease. These findings were further tested by in vitro methods where splenic and lymph node lymphocytes from infected hamsters were examined for their ability to respond to Con A in terms of the induction of antigen non-specific suppressor T cells. The mixing of Con A stimulated splenic or lymph node lymphocytes from infected hamsters was unable to inhibit the primary antibody responses of SRBC as compared to the normal control.(ABSTRACT TRUNCATED AT 400 WORDS)     

Proliferative response of T lymphocytes to a proline-rich polypeptide (PRP): PRP mimics mitogenic activity of Il-1

Mitogenic properties of a proline-rich polypeptide were investigated. The mitogenic action of PRP was compared with the mitogenic action of Il-1. PRP was not mitogenic for thymocytes at doses 0.01-50 micrograms/ml. PRP, at doses 0.1-50 micrograms/ml, augmented the proliferative response of thymocytes to Con A in a similar fashion as Il-1. At doses higher than 10 micrograms/ml, PRP induced proliferation of lymph node cells and splenocytes as well as T cells from the lymph nodes. It did not, however, cause significant proliferation of B cells from the lymph nodes, at the doses used. PRP did not induce proliferation of an antigen specific Lyt 1+ T cell clone. Il-1 behaved in a similar way as PRP in all the tests described. We consider a possibility that under physiological conditions, at a very early stage of postneonatal life, PRP may replace some functions of Il-1.