Mouse Thymus Cells Research Articles

Responsiveness of thymus cells to syngeneic and allogeneic lymphoid cells.

THE thymus is necessary for the normal development of cell-mediated immunity in mice as shown by the immunological defects after neonatal thymectomy1. Thymus cells themselves can be stimulated by allogeneic lymphoid cells in mixed leucocyte reaction (MLR)2 and become killer cells or cytotoxic lymphocytes after stimulation with allogeneic spleen cells in vitro (H. Wagner and M. Feldmann, unpublished work) and in vivo3,4. This suggests that the thymus as well as peripheral lymphoid tissues contain T cells which can be stimulated by foreign histocompatibility antigen to divide and differentiate into the cytotoxic lymphocytes which mediate cellular immunity. There have been suggestions that thymus cells might be stimulated to divide by “self” antigen, as well as foreign cells: incorporation of 3H-thymidine above background levels has been found in cultures with syngeneic spleen and thymus cells of adult rats5, although the experiments do not determine whether thymus or spleen cells have been stimulated. In contrast to these experiments, Howe et al. reported that only thymus cells of neonatal CBA mice reacted to allogeneic and syngeneic spleen cells of adult animals in “one way” MLR cultures6,7. Whether the reaction of neonatal thymus cells to syngeneic adult spleen cells is recognition of “self” antigens is uncertain, since spleens of adult mice could carry antigens which do not occur in neonatal animals and are therefore “unknown” for neonatal thymus cells. We demonstrate here that neonatal thymus cells do not react to 4-day-old CBA spleen cells, but adult thymus cells do react against both allogeneic and syngeneic adult spleen cells.

Antibody-inhibiting activity of bone marrow cells in vitro.

Mouse bone marrow and thymus cells have been studied according to their immunological reactivity. The test system involved measurement of the levels of IgM plaque-forming cells following the immunization of spleen cells <i>in vitro</i> in the presence of graded numbers of syngeneic bone marrow and/or thymus cells. All experimental evidence showed the existence of a radiosensitive subpopulation of cells in the bone marrow, capable of inhibiting both the primary and secondary IgM-antibody production irrespective of the dose of antigen used in the system. That this inhibition of antibody production may represent a central control mechanism was indicated by a small change in reactivity of antibody-inhibiting cells derived from the bone marrow of tolerant or normal animals. Furthermore, the inability of supernatants from bone marrow cells, premaintamed <i>in vitro</i> with or without antigen, to suppress the immune response suggested the absence of a released humoral factor responsible for their antibody-inhibiting activity. An enhancement of the immune response with all doses of thymus cells and their inability to overcome the inhibiting activity of bone marrow cells suggested that these immunological events are operating independently of each other.

Antigen-Binding Cells

Abstract Spleen, thymus and peritoneal cells of immunized mice were incubated with radioactive antigens and studied by autoradiography. A small number of lymphocytes in the spleen (0.1% to 0.5%) but very few in the thymus (0.01%) bind radioactive hemocyanin. The binding of antigen to lymphocytes apparently takes place by antibody receptors on their membranes. Exposure of most lymphocytes to antibodies to Ig blocked the binding. It was also found that the number of cells binding weak or strong immunogens was similar. Lymphocytes have been found that bind an autologous antigen, murine growth hormone. No lymphocytes bind mouse albumin. Incubation of spleen cells from immune or non-immune mice with highly radioactive KLH in vitro suppressed the capacity of the cells to adaptively transfer an immune response in x-irradiated mice. The inactivation of the adoptive immunity was specific since the treated cells made antibody to unrelated antigens. Macrophages took up radioactive antigen and this uptake was not blocked by exposure to anti-Ig.

Autoantibody-secreting plaque forming cells in spleen and thymus of NZB and normal mice.

NZB mice develop several manifestations of autoimmune disease and, in particular, Coombs positive haemolytic anaemia1 which can be accompanied by hepatosplenomegaly, glomerulonephritis and the appearance of antinuclear antibodies. The anaemia is associated with erythrocyte autoantibodies detected by direct and indirect antiglobulin tests and which from 3–4 months of age appear in an increasing proportion of mice until virtually all are involved by 9–12 months. Although there has been a good deal of work on the various disorders in NZB mice, the aetiology of the disease remains obscure2. The thymus has been suggested as the site of origin of the disease3 and recently a viral cause for the autoimmune state was proposed4, but this evidence for these hypotheses has been equivocal5–7. Although the spleen has been implicated as the chief site of autoantibody production8, no direct demonstration of the autoantibody secreting cells in NZB mice has been reported.

Limited synergism between rat thymus and mouse bone marrow.

IN a number of experimental systems1 it has been shown that thymus and bone marrow-derived cells can cooperate in the production of humoral antibodies. In some instances this synergism has been demonstrated when the interacting cells are antigenically similar but in other experiments allogeneic cells have been found to cooperate. We have now demonstrated cooperation between rat and mouse cells. A two-step experiment was performed, following the design of Miller2. Rat or mouse thymus cells were introduced with antigen into irradiated recipients. The donor cells were collected and injected into secondary host mice. The details of these experiments and their results are as follows.

In vitro stimulation of mouse thymus cells by PHA and allogeneic cells

The in vitro reactivity of mouse thymus cells to phytohaemagglutinin (PHA) and allogeneic cells has been investigated using the incorporation of tritiated thymidine as a marker for cellular stimulation. It was observed that thymus cells from untreated donors were stimulated to a very slight extent in the mixed leucocyte culture (MLC) and by PHA compared to cells from lymph nodes and spleen. However, highly reactive thymic cells could be obtained by treating the donor animals with cortisone acetate. This treatment also enhanced the reactivity of the animals' lymph node cells, but the increase was small relative to thymus cells. Cortisone-resistant thymic cells cultured together with allogeneic mitomycin-treated cells were found to be transformed into killer cells. These cells could specifically kill fibroblasts in vitro carrying the same major histocompatibility antigens as the mitomycin-treated cells. The results strongly suggest that the cells in the thymus which can be stimulated by PHA or allogeneic cells are part of the relatively cortisone-resistant fraction of the thymus cell population.

Kinetic differences in unresponsiveness of thymus and bone marrow cells.

Both thymus and bone marrow cells of adult mice can be made specifically unresponsive to human gamma globulin, but each cell population displays a distinct kinetic pattern for both the induction and spontaneous loss of the unresponsive state. These kinetics appear to be much slower in the bone marrow cells than in the thymus cells. In addition, the dose of deaggregated human gamma globulin needed to induce unresponsiveness in bone marrow cells is much greater than that needed to induce unresponsiveness in thymus cells. Apparently, unresponsiveness in only one cell type is sufficient for the tolerant state to be exhibited by the intact animal.

27. Resistance mechanisms of Friend virus induced leukemia

Immunological resistance against Friend virus seems to be primarily mediated vy antibody formation. No cell-mediated immune response against the virus was found in immunized mice. Besides immunological basis. Other mechanisms play a major role in resisteance and susceptibility. The possibility of a ’target cell“ capable of virus binding in the hematopoietic tissues of susceptible mice was studied by using a new tecnique. Known numbers of focus units (FFU) of Friend virus B (passaged in DBA 2 mice) were incubated with variable amounts of cells from different origins. After 30 min of incubation, the supernatant was injected intravenously in DBA/2 susceptible mice. Nine days later, the spleens were removed and the number of surface foci was counted after immersion in Bouin's fixative. The number of foci observed in these animals represents the number of FFU left in the supernatant after incubation. Controls were injected with unabsorbed virus kept for 30 min at room temperature. Using this method, we found that the Friend virus was adsorbed by spleen, bone marrow, or thymus cells of mice from susceptible strains, e.g., C31I or DBA/2, but was not adsorbed by other tissues from the same strains. By contrast, when spleen or bone marrow cells from resistant strains were incubated, e.g., C57BL/I, C57BL/6, C58, the virus was not adsorbed and the original number of FFU was obtained in the supernatant. These date strongly suggest the presence of a target cell in hematopoietic tissues of susceptible mice and its absence in resistean strains.

Stimulation of DNA Synthesis In Vitro in Mouse Thymic Cells by Bone Marrow Preparation

Abstract Some characteristics of a bone marrow factor were ascertained, on the basis of its ability to increase 3H-thymidine incorporation into thymic cells in vitro. It was found that the factor was not detectable in kidney or spleen preparations derived from the same mice from which active bone marrow preparations were obtained. The activity of the factor was reduced shortly after total-body irradiation of the mice, and increased again during the regeneration of the bone marrow. It is further shown here that the bone marrow factor has no effect on 3H-thymidine incorporation into mouse bone marrow cells. Leukemic mouse thymic cells show a significantly reduced response towards the bone marrow factor in comparison to normal thymic cells. The bone marrow factor has less effect on protein synthesis than on DNA synthesis in thymic cells, and its stimulatory effect on DNA synthesis is not dependent on protein synthesis. The isolated bone marrow factor is resistant towards heating at 100°C, even at a very low or very high pH. However, it is more labile to heat when in an impure state. Its activity is not affected by RNase, DNase, pronase and carboxypeptidase. It is insoluble in ether, but soluble in absolute ethanol.

Ontogeny of cellular immunity: Development in rat thymocytes of mixed lymphocyte reactivity to allogeneic and xenogeneic cells

The one-way mixed lymphocyte reaction of rat thymocytes to allogeneic cells was studied, using ACT and Fischer rat strains. Mitomycin-treated thymocytes or thymocytes from F 1-hybrid rats were used as the stimulating cells. The reactivity of neonatal rat thymus cells was somewhat lower than that of adult rat thymocytes, but was nevertheless quite significant. No reactivity could be determined in neonatal rat thymocytes to mitomycintreated xenogeneic (mouse) cells, although thymocytes from 7-day-old rats reacted equally well to allogeneic and xenogeneic cells. Graft-versus-host reactivity of both rat and mouse thymus cells was also determined. It was found that neonatal and adult rat thymocytes, as well as adult mouse thymocytes, could induce splenomegaly in neonatal mice, but not in neonatal rats. The implications of these findings are discussed in the light of recent theories linking reactivity to histocompatibility antigens to antibody formation.

Transplantation antigens in a high molecular weight form—II Non H-2 mouse antigens and membrane structure

Mouse thymus cell membrane preparations were treated with sodium dodecyl-sulphate and starch stearate and the soluble fraction was examined on DEAE Sephadex. One component carried H-2 antigen with LyA. LyB and theta antigens: other components carried theta antigen only. TL antigens were present but could not be exactly located in relation to the other alloantigens. The preparations are discussed in the light of membrane structure.

Cell‐mediated immune response in vitro: Independent differentiation of thymocytes into cytotoxic lymphocytes

AbstractDifferentiation of dispersed mouse thymus cells into cytotoxic lymphocytes has been shown for the first time to occur in an in vitro allograft system. It is concluded that the generation of cell‐mediated cytotoxicity is a T cell phenomenon with no thymus‐bone marrow synergism being required.

Antigen-binding cells in normal mouse thymus.

The thymus of a normal adult mouse contains lymphocytic cells with a large number of antigen-binding receptors. Binding of the antigen by these cells is specific and can be inhibited by cross-reactive materials. It is possible that the interaction of thymocytes with antibody-forming precursor cells, required in the primary immune response to certain antigens, is mediated by these specific antigen-binding cells of the thymus.

Specific cytotoxicity by sensitized mouse thymus cells on tissue culture target cells

Specific in vitro cytotoxicity by mouse thymus cells on allogeneic tissue culture target cells was investigated employing a microassay. Lymph node and spleen cells from mice immunized against the target cells were highly active in the test, while thymus cells showed no appreciable effect. In contrast, a strong reactivity of thymus-derived cells was detected following intravenous transfer into irradiated allogeneic and F 1 mice. This graft-versus-host immunization was detected by testing spleen cells from the host in an appropriate immune cell-target cell combination. The specific donor-versus-recipient direction of sensitization indicated that cells derived from the thymus were responsible for the effect. Moreover, the proportion of these reactive cells in the thymus was enriched by cortisone treatment of the donor animals. It is concluded that a population of lymphocytes in the thymus or their descendants which are relatively cortisone resistant and probably located in the medulla, are competent in mediating cellular immunity.

Characteristics of the immunocompetent cells in the mouse thymus: Cell population changes during cortisone-induced atrophy and subsequent regeneration

The cellular composition and the immunological reactivity of the mouse thymus were studied during the involution and regeneration induced by cortisone. It was found that the majority of the thymus cells that disappeared during the first 2 days after cortisone treatment were immunologically inert, since the total immunological reactivity of the organ was almost unaffected despite the fact that the total cell number was reduced to 3%. The remaining immunocompetent cells exhibited a larger average cell volume than did the bulk of the thymus cells. They were also relatively radioresistant as compared to the thymus cells of untreated mice. The immunological reactivity was measured by a graft-versus-host test and by the capacity of the cells to restore humoral antibody production of thymectomized, irradiated, and bone marrow reconstituted mice. During the early regenerative phase the relative immunological competence and the proportion of radioresistant thymus cells decreased in parallel. During this time period the volume of the cells increased. Regeneration was completed around day 14 after cortisone treatment when both the cellular composition and the immunological reactivity were normalized.

Cellular recognition by mouse lymphocytes in vitro. I. Definition of a new technique and results of stimulation by phytohemagglutinin and specific antigens.

The media and culture conditions required for in vitro stimulation of mouse lymphoid cells are described. The medium was arginine-rich and contained heat-inactivated human serum. A component of the human sera necessary for stimulation of the cells was a natural mouse cell agglutinin, which affected both background stimulation and the degree of induced stimulation with phytohemagglutinin (PHA). Absorption of the agglutinin from the human serum rendered the medium incapable of sustaining DNA synthesis in the presence of PHA. The response to PHA of mouse spleen and thymus cells was age-dependent and, although this response was not present at birth, it rapidly rose to adult levels. Spleen cells from mice immunized with bacillus Calmette-Guérin (BCG) or sheep erythrocytes (SRBC) showed increased in vitro reactivity to added purified protein derivative (PPD) or SRBC stroma, dependent on the time of immunization. The dose response curve for the SRBC stroma stimulated, immune spleen cells is compatible with a theory of cell to cell interaction being necessary for an in vitro reaction to antigen. The possible role of the mouse cell agglutinin (AMLG) is discussed.

Anti-theta antibodies for detecting thymus-dependent lymphocytes in the immune response of mice to SRBC.

MOUSE thymus cells possess a high concentration of the theta (θ)1 and Ly2 antigens. Several approaches have demonstrated that the sensitivity of peripheral muriiie lymphocytes to the cytotoxic effect of θ and Ly antibodies is a thymus-dependent property. The population of cells sensitive to the cytotoxic effect of θ antibodies disappears from lymph nodes3 and spleen4 after neonatal thymectomy. Similarly, following neonatal thymectomy, the population of cells sensitive to Ly antibodies disappears from lymph nodes (unpublished results of M. S. and Yron). Following administration of heterologous anti-lymphocyte serum, a procedure resulting in elimination of thymus-dependent lymphocytes5,6, cells sensitive to θ antibodies disappear transiently from the lymph nodes7 and spleen4.

A comparison of the immunologic function of thymus cells at varying stages of maturation.

SummaryInjections of mouse thymus cells from fetal, neonatal, 4-week- and 1-year-old syngeneic donors were compared for their ability to potentiate the production of serum antibody to sheep red blood cells in thymectomized, irradiated recipients previously injected with syngeneic bone marrow. Five million fetal thymus cells afforded no restoration of the immunologic response and one-year-old thymus cells gave marginal improvement. The apparent deficiency of the fetal thymus cell inoculum was quantitative since 20 million of these cells proved to be as active as five million neonatal thymus cells. Neonatal and 4-week-old thymus cells induced significant and virtually equal immunologic reactivity in terms of serum antibody titers. Immunologic restoration estimated by the number of spleen direct PFC was insignificant in all the groups given thymus cells. There was no increase in the number of indirect PFC observed in the experimental groups except for the one treated with 4-week-old thymus cells and here the...

Assay for the Immunosuppressive Capacity of Antilymphocyte Serum

Abstract In this and the preceding two papers an assay procedure for ALS has been described. It measures the capacity of ALS to opsonize either mouse, human or dog thymus cells. The percentage of mouse thymus cells opsonized by an anti-mouse lymphocyte serum correlates with its ability to depress the immune response of mice to SRBC. Based on this finding, it is postulated that anti-dog or anti-human lymphocyte sera that are capable of opsonizing dog and human thymus cells, respectively, will be immunosuppressive.

Dialysable agent involved inthe stimulation of thymic regeneration following injury.

WE have found with in vitro and in vivo test systems a dialysable agent in the bone marrow of mice and rabbits which stimulates proliferation of mouse thymic cells. The in vitro test was based on 3H-thymidine incorporation into DNA of thymic cells, and the in vivo test on regeneration of X-ray injured thymuses.