Mouse Thymus Cells Research Articles

The detection of in vitro monocyte‐macrophage colony‐forming cells in mouse thymus and lymph nodes

In vitro monocyte-macrophage colony-forming cells (CFC) have been detected in the thymus (30/10(6) cells) and in the cervical (22/10(6)) and mesenteric (20/10(6)) lymph nodes (LN) of the mouse. Thymus and LN derived CFC differed from bone marrow derived CFU-c in several characteristics parameters: (1) sole specificity of PMUE to induce colony formation (CF), (2) apparent singular line of monocyte-macrophage differentiation, (3) a marked 6- to 10-day lag period prior to initiation of CF, and (4) significantly slower rates of appearance of colonies in culture after initiation of CF. Two of these parameters are shared with those CFC detected within alveolar space, peritoneal exudate and pleural effusion. These are the delay prior to CF and the singular monocyte-macrophage differentiation. These similarities suggested that T-CFC and LN-CFC are probably of similar origin and represent, as suggested by Lin and Stewart ('74), a population of progenitor cells exclusively for monocyte-macrophages.

Generation of cytotoxic effector cells by immunocompetent thymus cell subpopulations.

Immunocompetent mouse thymus cell subsets (TH-2) cultured with allogeneic TH-2 cells, required a peripheral lymphoid cell in order to generate specifically cytotoxic T cells (CTL) in vitro. The helper cell is identified as a B cell, and can be supplied either in the mixture of target or responding peripheral cells, or in optimal ratios of 10:1 to 20:1 as nonproliferating cells syngeneic to the responder, when both responder and target are TH-2. Allogeneic helper cells are much less effective. Kinetic studies indicate that helper cell activity involves initial interaction with stimulator cell and is then required throughout the period of generation of cytotoxic lymphocytes. Addition of critical levels of certain IgG preparations, particularly IgG2A appears to support generation of CTL in TH-2/TH-2 cultures but is considerably less effective than intact B cells. Cytotoxicity generated under these conditions has the same genetic requirements for I-region histoincompatibility between stimulator and responder, and for K or D identity of stimulator and target cell, as has been reported for peripheral cell CTL.

FETAL SUPPRESSOR CELLS

The cell-mediated immune responses are significantly reduced in fetal and newborn mice, furthermore, spleen cells of these animals are able to suppress the immune responses of adult lymphocytes in a variety of situations. Newborn mice sensitized to picryl chloride within 24 hr after birth fail to develop contact sensitivity reaction when tested several weeks later, and fetal mice do not develop graft-versus-host reaction when given injections of parental lymphocytes. Spleen cells of fetal or newborn mice are able to suppress the passive transfer of contact sensitivity and the local graft-versus-host reaction elicited by immunized parental cells in F1 hybrid mice, and reduce significantly the severity of graft-versus-host reaction and mortality rate in cyclophosphamide-treated F1 recipients. In no experiment were thymus cells of either fetal or newborn mice found to be inhibitory. The possible mechanisms of action and biological significance of fetal suppressor cells are discussed.

A large glycoprotein of moloney leukemia virus derived from interferon-treated cells

Moloney murine leukemia virus produced in interferon-treated mouse thymus and bone marrow cells has a high particle to infectivity ratio (1, 2). This virus contains a prominent glycoprotein with a molecular weight of about 85,000. This large glycoprotein is only a very minor component of Moloney leukemia virus produced in control TB cells and may be an uncleaved precursor to gp 69–71 (3, 4).

The heterocytotoxicity of human serum: I. Activation of the alternative complement pathway by heterologous target cells

Human serum induces cytolysis of mouse thymus and thymoma cells, and cytostasis of mouse bone marrow and spleen cells, and various methylcholanthrene-induced tumour cells. The latter was manifested by deficient metabolic activity when cultured in the presence of fresh human sera. Decomplementation procedures demonstrated that these heterocytotoxic effects are mediated in part via activation of the alternative complement pathway in human serum samples. The presence of properdin and C3 on the target cell surface was confirmed by immune adherence and indirect immunofluorescent tests. Activation of the alternative complement pathway was elicited by incubation of the human serum with the relevant target cells, resulting in the appearance of the cathodal migrating fragment of the factor B, denoting complement activation. The following publication will present evidence that activation of the alternative complement pathway takes place via an antibody-independent mechanism acting at the cell surface. These and other observations in the literature raise the possibility that activation of the alternative complement pathway by surface cell receptors on tumour cells represents a mechanism of natural immunity versus tumours.

The effect of papaverine on the fate of thymidine phosphates in isolated mouse thymus cells

The burst of incorporation of 3H into DNA of mouse thymocytes during an incubation at 37° for 5 min. following a preincubation at 4° for 30 min. is markedly inhibited by papaverine (0.1 mM). This event is accompanied by an efflux of 3H into the medium, largely in the form of thymidine. No enhanced efflux of 3H is detected when DNA synthesis is blocked by hydroxyurea (1 mM). While it is uncertain that papaverine has a separate effect on DNA synthesis, the reduced incorporation into DNA could be explained by its ability to increase the breakdown of intracellular thymidine phosphates.

Functional Maturation of Mouse Thymus Cells

SummaryOntogeny of thymus cell function in humoral antibody response was studied in vitro using cells from different embryonic stages and neonates of BDF1 mice. As early as the sixteenth day of embryonic development, the thymus contained cells that were readily activated in vitro by con A to function as helper cells in an antibody response against sheep erythrocytes. The helper function of embryonic thymocytes increased rapidly, reaching adult levels before birth. It has also been shown that con A-treated thymus cells are less susceptible to anti-O serum and correspondingly more susceptible to anti-H-2 serum; the implication of this finding in thymus cell maturation has been discussed.The author is grateful to Professor Robert Auerbach for his encouragement and criticisms during the course of this work. This study was supported in part by Research Grant CA 13548 from the National Institute of Health.

The differentiation of T-lymphocytes: III. The behaviour of subpopulations of mouse thymus cells in short-term cell culture

Adult mouse thymus cells were incubated at 37 °C in culture medium in order to examine various aspects of T-cell differentiation. The level of dividing cells fell rapidly after culture, and there was a reduction in cell size. A striking effect was the loss of thymocyte viability which occurred in two phases. A total lysis of some cells occurred over the first 2 hr, but this was less extensive with higher cell concentrations, or in the presence of serum. This phase was followed over the next 70 hr by a steady loss of cell viability, even under optimised conditions. Thymocyte death was more extensive than the death of spleen or lymph node cells under the same conditions. Thymocyte death in culture was limited to a particular subset of cells. Rapid loss of viability was a constant feature only of the major thymocyte population (characterised as high Thy 1 alloantigen (θ), low histocompatibility antigen (H2), thymus leukemia alloantigen (TL) + ve, and cortisone sensitive). Within this group survival time was a function of intrathymic age, with maximum lability in older cells. Cells initially large survived better than small cells. In contrast, the minor thymocyte subpopulation (characterised as low θ, high H2, TL −ve, and cortisone resistant) survived well, with an occasional increase after 5–24 hr of culture. Isolated populations of low-θ cells gave the same good survival in culture, showing they were not simply being replenished from the high-θ elements. The results document another basic difference between the major (high θ) and minor (low θ) thymocyte subpopulations, namely, differential survival in cell culture. The results are in line with the view that the major and minor subpopulations represent largely independent lines of development, rather than having direct precursor-product relationship. The extreme lability of the major, high-θ subset could reflect a programming for intrathymic death.

Factor producing lysis of thymus-derived cells in vitro by specific antigen

Lymph node cells of immunized mice were lysed by specific antigen in vitro. These cells were not lysed by antigen after washing them four times with Hanks' solution. The factor lysing lymph node cells and thymus cells of normal mice in the presence of the specific antigen was found in the supernatant of the lymph node cell suspension of immunized mice. Bone marrow cells were also lysed by the factor and specific antigen only after preincubating the cells in extracts of thymus or lymph nodes of normal mice. When active supernatants were fractionated by gel filtration on Sephadex G-200, the active material eluted with molecules of approximately 20,000–45,000 MW. This factor was homogeneous on electrophoreses in polyacrylamide gels and detected in the prealbumin zone.

Colony-forming ability of the bone marrow in mice after burn trauma

The dynamics of the number of colony-forming units (CFU) in the bone marrow of CBA mice after receiving third-degree thermal burns covering 15% of the body surface was studied by the exogenous splenic colony method. The number of CFU in the bone marrow of the mice was reduced by 41–52% on the 4th and 16th days after burning. Thymus cells of intact mice, if injected simultaneously with bone marrow of the burned mice, increased the number of exogenous splenic colonies formed in the recipients. The results suggest that not only is the number of CFU reduced in the bone marrow after burns, but also the number of thymus-dependent cells necessary for normal colony formation.

Studies on Human Brain-Thymus Cross-Reactive Antigens

Antigens shared by human brain and thymocytes and by human and mouse tissues were studied with rabbit anti-human thymocyte antiserum (RAHT). It was found that cytotoxicity of RAHT serum against mouse thymus cells was not absorbed by mouse liver or bone marrow cells. Human brain and thymus cells completely absorbed the anti-thymocyte activity from this antiserum. It was suggested that human brain had antigenic determinants identical or very similar to those found on human thymocytes. Activity of RAHT antiserum against mouse thymus cells was completely removed by an absorption of mouse brain and thymocytes. These results demonstrated that there were shared antigenic determinants between human and mouse tissues.

Different Mechanisms for the Modulation of TL Antigens on Murine Lymphoid Cells

An indirect radioimmunoassay has been used to study the antibody-induced changes in expression of TL antigens on mouse thymus and leukemia cells. The results obtained indicate that the incubation of TL+ cells in relatively high concentrations of anti-TL antisera results in a detectable, but not major, loss in the quantity of antigen detected in the plasma membranes. This decrease is not inhibited by azide. Although the antigen loss seems to coincide with the appearance of patching of membrane TL antigens on leukemia cells observed in indirect immunofluorescence microscopy, this is not the case when thymocytes are used as targets. Fab fragments prepared from anti-TL antibodies can also induce a quantitative decrease in antigen expression. The major mechanism involved in membrane antigen loss seems to be endocytosis, although some shedding may also occur. Resistance to immune cytolysis (antigenic modulation) occurs during incubation in anti-TL antibodies before either endocytosis or antigen redistribution has progressed enough to be directly responsible. Since antigenic modulation does not represent a depletion in TL antigens being expressed on the cell surfaces, another mechanism must account for the failure of guinea pig complement to achieve cell lysis.

Cross-reacting thymus and brain antigens in the cerebral cortex

Rabbit antisera against antigens of whole mouse, rabbit, guinea pig, and human brain were found to have a cross cytotoxic action on lymphocytes of the thymus, lymph nodes, and spleen of the animals of these species. Mouse thymus cells were the most sensitive (index of cytotoxicity 63–100%); the cells of other mouse lymphoid organs and lymphocytes of the other species of animals and man were more resistant. Bone marrow cells were not injured by any serum. Antigens responsible for the cytotoxic properties of the sera were found to be located in the human cerebral cortex and to be absent from the white matter and the brain stem.

Cytotoxic and complement-fixing activity of rabbit antisera against rat and human brain cortex and white matter

The cytotoxic and complement-fixing activity of antisera against the cortex and white matter of the rat and human brain was investigated on thymus and bone marrow cells of mice and rats. The cytotoxicity test proved to be more sensitive and accurate and showed that cytotoxins against rodent thymocytes were present only in antisera against the cortex of the human brain but they were present in antisera against both the cortex and the white matter of the rat brain, although in significantly larger amounts in the former. Sera against rat cerebral cortex, when exhausted by the homonymous antigen, lost their cytotoxicity but they retained it after exhaustion with white matter.

Suppression of Antibody Responses in Allogeneic Mice by Products of Lymphoid Tissue

Abstract A cellfree extract prepared from the spleen cells of C3H mice is capable of suppressing antibody responses to SRBC when extract material is exposed to alloantigens. The observed immunosuppression was attributed to a soluble factor in the extract. This allogeneic suppressive factor (ASF) was detected in extracts prepared from the spleen cells of unirradiated mice as well as those of irradiated mice repopulated with thymocytes, provided that mice were previously immunized with SRBC. Donors of actively suppressive ASF preparations did not need to be previously exposed to alloantigens. Extracts from thymus and marrow cells of unirradiated mice and the spleen cells of irradiated mice repopulated with marrow cells (or no cells) did not contain ASF. C3H thymocytes stimulated with SRBC generated more ASF activity in spleens of C3BF1 hosts than in those of C3H hosts, indicating that alloantigenic stimulation enhances the production or activity of ASF. Once produced, C3H ASF was able to suppress antibody responses in cell transfer experiments only if exposed to C3BF1 alloantigens of either donor lymphoid cells or irradiated hosts. Once exposed to alloantigens, ASF appears to be capable of suppressing antibody responses of syngeneic C3H or semi-allogeneic C3BF1 cells. When both donor lymphoid cells and hosts were syngeneic with the donor of the ASF, there was enhancement of antibody formation in cell transfer experiments. C3H ASF did not interfere with education of C3BF1 thymocytes to SRBC or with the generation of precusors of anti-SRBC antibody-forming cells by C3BF1 marrow cells. ASF may interfere with cellular cooperative events necessary for humoral immune responses or with terminal differentiation of B cells. Production of ASF could partially account for the suppression of antibody responses observed during graft-vs-host reactions.

Evidences for a “New” C1 Inhibitor, Factor S, Released from Leucocytes

Abstract Pretreatment of sheep erythrocytes (E) with supernatants of short-term cultures of mouse spleen or thymus cells induces the following interactions with guinea pig complement: 1) Supernatant pretreated EAlim (S.EAlim) are less susceptible to lysis by excess complement. S.EAC1̄4 yield a lower C2 titer, have an increased Tmax, but an unchanged C2 decay when compared with medium-pretreated EAC1̄4. Lysis of already formed EAC1̄4̄2̄is not inhibited. Therefore the inhibition affects C2 activation. 2) E, pretreated with supernatant (S.E), are able to fix C1̄, as detected by transfer to EAC4: In order to generate the same number of C1̄-fixing sites by using IgM hemolysins, five to six times the optimal amount is required. Yet S.EC1̄ cannot be lysed by the other components added in excess. S.EC1̄ can remove C4 from the fluid phase with the same efficiency as EIgMC1̄ but S.EC1̄ treated with C4 cannot remove C2. 3) Native C1̄ binds to S.E: S.E and EIgM remove C1 from the fluid phase with the same efficiency; fixation of 1̄ on S.E is decreased after incubation with native C1̄

Precursor cells of fibroblasts detected by in vitro cloning of cells from hematopoietic organs of normal and irradiated mice

Colonies of fibroblasts are formed in monolayer cultures of bone marrow, spleen, and thymus cells of adult mice with an efficiency of colony formation (per 105 cells) of 2.2 for bone marrow, 0.20 for spleen, and 0.16 for thymus. On irradiation of mice with a dose of 150 R, about half of the fibroblasts colony-forming units in the bone marrow die; during the next 6 days their number falls a further fivefold, with a return to the normal level 25 days after irradiation.

Preparation and Effects of an Anti-B Cell Serum

An heterologous antiserum specific for bone marrow-derived cells (B cells) was prepared by immunizing rabbits with lymph node cells from nude mice. After absorption with mouse red blood and thymus cells, the antiserum killed a population of cells from various lymphoid organs and the cytotoxic effects were inversely related to those of anti-theta antibody. When bone marrow or spleen cells were treated with the antiserum and guinea pig complement before transfer into irradiated mice, the number of plaque-forming cells was greatly reduced in the spleen of the recipient. Pretreatment of thymus cells with the antiserum in a similar way resulted in no inhibition of hemolytic plaque. When spleen cells from mice previously immunized with sheep red blood cells were treated with the antiserum and complement, the formation of hemolytic plaque was not affected. These findings indicated that the antiserum was specific for B cells and that the number of antigenic determinants on B cells to which the antiserum reacted decreases during differentiation into antibody-forming cells.

Complement inhibitor(s) released from leukocytes: II. Evidence that lymphocytes release and produce an inhibitor of C2 activation

We have previously reported that mouse spleen and thymus cells in short term culture release an hitherto undescribed complement inhibitor that prevents the activation of C2. Here it is shown that the inhibitor is released by B lymphocytes and may be produced by lymphocytes. Release of the inhibitor takes place at 37 °C and in the presence of metabolic inhibitors, but not at 4 °C. Thus the release of the inhibitor could be related to the shedding of structures located on the lymphocyte membrane.

Cholera Toxin and the Adenylate Cyclase-Activating Signal

Studies with chemically modified cholera toxin derivatives showed that all treatments that decreased the ability of toxin to bind to mouse thymus cells or to polystyrene-coupled GM1 ganglioside caused a concomitant reduction in the toxin's ability to increase adenosine 3':5'-cyclic phosphate (cyclic AMP) in thymus cells and skin vascular permeability in rabbits. Dissociation of the H (heavy) and L (light) subunits abolished the biologic activity without inhibiting receptor binding, as did treatment with arginyl-specific reagents (which did not change the aggregation state of the toxin). When thymus cells were incubated with 125I-labelled toxin at 37C,only about 1% of the total cell-bound radioactivity was recovered in the cytosol supernate. Similar values were found for cells incubated with toxin at 0C, and with 125I-labelled choleragenoid at 37C or 0C. Thymus cells rapidly bound less than or equal to equal to 5 X 10(4) cholera toxin molecules per cell at both 0C and 37C. Much less, however, of the radioactive toxin bound at 37C than of that bound at 0C was displaced by addition of unlabelled toxin or choleragenoid. Similar temperature-related irreversible binding was noted with 125I-labelled choleragenoid. The relative amounts of H and L subunits in the irreversibly cell-bound and in the displaced 125I-labelled toxin were indistinguishable. Treatment of thymus cells at 37C, but not at 0C, with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide caused a 10-fold reduction of adenylate cyclase stimulation by cholera toxin without inhibiting activation by epinephrine or prostaglandin E1, or appreciably altering the basal, unstimulated enzyme activity. The carbodiimide inhibited the cyclic AMP response to cholera toxin when added shortly after the toxin had bound to the cells (early in the lag phase).