Spleens Of Irradiated Mice Research Articles

Inhibition of erythroid cell growth in irradiated mice by allogeneic lymphoid cells: Specificity of the response

Allogeneic lymphocytes can inhibit proliferation of erythroid cells in the spleens of irradiated mice grafted with syngeneic bone marrow cells. Since there is a linear relationship between the number of injected lymphocytes and erythroid activity, the method is frequently used as a graft-vs-host assay. In this investigation we show that the reduction of erythroid activity is due to both a reaction of the lymphocytes against the tissues of the host and against the grafted bone marrow cells. Thus, reduced erythropoietic activity does not necessarily indicate that the bone marrow targets possess antigens against which the lymphocytes are reactive.

The Expression on Mouse Lymphoid Cells of Th-B, an Antigen Common to Mouse B Cells and Thymus Cells

Abstract A new cell-surface antigen of mouse lymphocytes, which was found earlier on normal and malignant plasma cells and about half the normal thymus cell population, has been shown to be on mature B cells and on the least immunocompetent subpopulation of cells of the thymus. The antigen was first detected by the cytotoxic effect on relevant cells of an in vivo purified rabbit antiserum raised against cells of the mouse IgM-producing plasma cell tumor MOPC-104E. We have now used a cell-transfer method to examine the effect of this antiserum (RantiM104E) on immunocompetent lymphocytes of spleen, including separated splenic B and T cells and thymus and bone marrow cells. We have found the antigen on the immunocompetent B lymphocytes of the spleen, but not on any other of the immunocompetent cells examined. The method involves determining the effect of the antiserum on the development of plaque-forming cells in the spleens of irradiated mice that have received lymphocytes from immune or non-immune donors by treating the lymphocytes with RantiM104E antiserum before transfer. Plaque-forming cells are suppressed when antiserum-treated spleen cells are transferred to the irradiated mice. This suppression was found to be due to action on only the splenic B cells. The helper activity of splenic T cells was not reduced significantly by antiserum treatment. Neither was the helper activity of thymus cells similarly treated reduced by the antiserum. We found, in fact, that the subpopulation of the thymus cells which is resistant to the antiserum was more immunocompetent than the total thymus cell population. In addition, the hydrocortisone-resistant thymus cells were also found to be resistant to the cytotoxic action of the antiserum. The antiserum had no effect on bone marrow cells in the cell-transfer procedure. The antigen involved, which we are designating “Th-B” appears to be a B cell line marker which appears relatively early during the differentiation of mouse B cells from precursor cells and is lost during maturation of T cells.

Inhibition of erythroid cell growth by allogeneic murine lymphocytes: Evidence for a synergism between lymph node cells and thymocytes

Murine lymphoid cells from thymus and lymph nodes were tested for synergistic response in a graft-vs-host test. The test is based on the principle that allogeneic lymphocytes inhibit erythroid cell proliferation in the spleens of irradiated mice infused with syngeneic bone marrow cells. I was observed that mixtures of thymocytes and lymph node cells from the same parental strain yielded graft-vs-host responses in irradiated F 1-hybrids higher than expected by summing the responses of the two cell populations tested separately. A similar synergistic response was obtained using mixtures of thymocytes and lymph node cells obtained from the two parental strains of the hybrid, whereas such an effect was not detected using mixtures of lymph node cells or mixtures of thymocytes from the two parental strains. Nor could synergy be demonstrated between parental strain lymph node cells and thymocytes syngeneic with the bone marrow target cells. Thymocytes obtained from one parental strain which were injected into its irradiated F 1-hybrid transformed into a population of sensitized cells in the spleens of the recipients. This transformation was suppressed by the simultaneous injection of lymph node cells from the second parental strain. Since there is a synergistic immune response by such cell mixtures it is concluded that thymocytes may enhance the graft-vs-host response of lymph node cells. Parental strain thymocytes and lymph node cells, the latter being specifically immunologically tolerant to the bone marrow target cells, failed to give a synergistic response indicating that thymocytes do not transform unresponsive lymphocytes into responsive, but rather enhance the reactivity of existing, specifically responsive cells. The results thus show that thymocytes may enhance the response of lymph node cells in this specific graft-vs-host assay.

Comparison of Irradiated and Mitomycin-Treated Mouse Spleen Cells As Stimulating Cells in Mixed Lymphocyte Cultures and in vitro Sensitization

Abstract Mitomycin-treated and irradiated mouse spleen cells were compared as stimulating cells in allogeneic mixed lymphocyte culture (MLC) and as sensitizing cells in in vitro allogeneic sensitization. C57BL/6 and BALB/C spleen cells were irradiated at doses up to 5000 rads or treated with mitomycin C to inhibit DNA synthesis. Irradiated cells, as well as mitomycin-treated cells, were efficient both in stimulating MLC and in sensitizing in vitro.

The Long-Term Effects of X-Irradiation on the Ability of the Spleen to Support Clonogenic Proliferation

A modified spleen colony method was used to measure the functional ability of the irradiated spleen to support clonogenic proliferation. This was assayed by counting the number of exogenous colonies formed by normal bone marrow cells injected into recipient mice whose spleens had previously been irradiated. A shallow dose-related decrease in the ability of irradiated mouse spleen to support clonal growth was seen at all times after splenic irradiation. These dose-response relationships do not resemble the usual clonogenic survival curves. The reduction in colony number produced by normal bone marrow cells did not decrease below 0.4 even when the recipient spleen had received as much as 3000 rads. Recuperation of the ability of irradiated mouse spleen to support clonal growth was observed at intervals of 6 and 9 mo. This appeared to be a temporary recuperation, since a second reduction was seen at 12 mo. An absence of repair was demonstrated when 2000 rads was fractionated into two doses delivered 6 hr to ...

Interferonogenicity and antigen recognition.

NOT only viral and syngeneic polynucleotides but also bacterial endotoxins, PHA (refs 1 and 2), heterologous and, in certain cases, homologous gammaglobuliris can induce interferon3–5. The question arises whether or not antigenicity correlates with interferonogehic properties. We therefore studied the interferonogenic properties of syngeneic and allogeneic living or irradiated mouse spleen cells (SC), xenogeneic and allogeneic erythrocytes and syngeneic materials (erythrocytes and sera) that had become autoantigenic after treatment with trypsin, iodination or heating.

Analysis of anti-hapten foci in repopulated mouse spleens.

AbstractWe have demonstrated by two separate techniques the formation in vivo of foci of anti‐(4‐hydroxy‐3‐iodo‐5‐nitrophenyl)acetyl (NIP) antibodyforming cells. The initiation and expansion of such foci in the spleens of irradiated mice has been shown to depend on the presence of both hapten‐and carrier‐primed spleen cell populations.Sensitive methods have been developed to assay the heterogeneity of antibody produced in single anti‐hapten foci, usually of less than 1000 plaque‐forming cells. Foci from NIP‐BSA (bovine serum albumin)‐primed spleen cell inocula have been shown to be usually monoclonal. Using mixtures of BSA‐primed and NIP‐ovalbumin primed spleen cells, the effect of changing the ratio of cooperating cell types upon the homogeneity of antibody in foci has been studied. The results are interpreted as preliminary evidence in favor of a high degree of B cell selection operating during focus initiation.An effect of carrier‐primed cell numbers on the size of anti‐hapten foci has been detected and some morphological features of foci are described.

Hemopoietic stem cell growth and microdiffusion

Extensive experiments carried out to study the kinetics of stem cell growth in the spleen of irradiated mice are reported here and the methods used are elaborated. The temporal and spatial features of the growth of the hypernetted, interacting system of stem cell chains in the spleen may be understood on the basis of a random distribution of source cells and of a microdiffusing stimulus for stem cell renewal along the cell chains. To test the validity of these assumptions, the growth kinetics are studied after administering various doses of colchicine to stem cells during different stages of growth. The behavior of the various phases of the recovery curves lends support to the theory of microdiffusion. Using the number of stem cells at saturation, the diffusion coefficient of the stimulus for renewal is approximately 10 -7 cm 2 sec -1.

Effect of blocking the pituitary-adrenal system on composition of endogenous colonies of hematopoietic tissue in the spleen of irradiated mice

Effect of blocking the pituitary-adrenal system on composition of endogenous colonies of hematopoietic tissue in the spleen of irradiated mice

Inhibition of erythropoiesis in the spleens of irradiated mice injected with allogeneic lymphocytes: Reactivity of lymphocytes against non-H-2 transplantation antigens

A sensitive technique has been applied for studying the graft-vs-host reactivity of murine lymphocytes against non-H-2 transplantation antigens. The method is based on the finding that allogeneic lymphoid cells are able to depress the growth of erythroid cells in the spleens of lethally irradiated mice injected with syngeneic bone marrow cells. Using this technique, it was possible to show that large numbers of normal, non-sensitized lymphocytes can depress the hematopoietic activity of bone marrow cells with different non-H-2 antigens. This effect was markedly enhanced by prior sensitization of the cells in the spleens of X-irradiated recipients having the same genotoype as the target bone marrow cells. Such sensitized cells did not exhibit any detectable reactivity against bone marrow cells having completely different transplantation antigens. They also exhibited an impaired capacity to react against bone marrow cells bearing both foreign H-2 antigens and the non-H-2 antigens against which the cells had been sensitized. Lymphoid cells which were recovered from the spleens of irradiated mice differing at both H-2 and non-H-2 transplantation antigens exhibited a strong reactivity against the foreign H-2 antigens but largely lacked reactivity against the non-H-2 antigens. The results emphasize that the present technique is useful for studies on the graft-versus-host reactivity of lymphocytes against weak transplantation antigens.

The persistence of hemopoietic stem cells in vitro.

Cells capable of forming colonies in spleens of irradiated mice (CFU) are lost temporarily when bone marrow cells from rats or mice are maintained in culture. Rat marrow CFU go through a minimum at about 3 days after which there is a slow increase in the number of CFU in culture, reaching a maximum at 9 days. Mouse marrow CFU reach a minimum at 3 days and a maximum at 7 days. Some rat marrow CFU persist in culture for as long as 28 days.

Differential Sensitivity of Bone Marrow and Thymus Cells to Actinomycin D in the in Vivo Response of Mice to Sheep Erythrocytes

Abstract Three cell types required for the hemolytic antibody response have been defined based upon their differential sensitivities to treatment with actinomycin D: a) the thymus-derived cell that is insensitive to treatment with 1.0 µg/ml of actinomycin; b) the antibody-forming precursor cell, of bone marrow origin, that is inhibited by 0.1 to 1.0 µg/ml of actinomycin and c) an auxiliary cell that is extremely sensitive to treatment with 0.01 µg/ml of actinomycin. Utilizing these sensitivities to actinomycin we determined the repopulation of each of the three cell types in the irradiated mouse spleen.

Amplification of B cell clones forming antibody to the 2,4-dinitrophenyl group.

AbstractThe selection of several single cell clones forming anti‐2,4‐dinitrophenyl (DNP) antibody in mice enabled us to study the life span of B memory cells, and to estimate the number of DNP‐binding cells belonging to a single clone, as well as their amplification into anti‐DNP plaque‐forming cells.B memory cells belonging to clone E9 were found to survive for several weeks in the absence of antigen. Memory cells for this clone were transferred into recipient mice; antigen challenge after 2 or 4 weeks led to E9 anti‐DNP formation at the same or half the level resulting from immediate antigen challenge.DNP‐binding cells were labeled with 125I‐DNP on a heterologous carrier protein (Maia Squinudo hemocyanin); for 3 different DNP‐clones the incidence of DNP‐binding cells in spleen (2nd transfer generation, 8 weeks after cell transfer) was 1000 – 1900 per 106 cells. This compares with 900/106 spleen cells in a multiclonal mouse primed with DNP‐ovalbumin; a proportion of these cells may represent T cells with specificity for DNP.Anti‐DNP‐forming cells in spleens of irradiated mice, receiving clonal memory cells and antigen, appeared after a lag of 4 – 5 days, reached a peak of 12000 – 18000 PFC between day 8 and 11 and then declined rapidly despite continued high levels of antibody production in recipient mice; this suggests antibody formation at sites other than spleen. The data are consistent with around 1 % effective homing of B precursor cells in spleen.

Effect of nonsyngeneic lymphocytes on differentiation of stem cells in the spleen of irradiated mice

Effect of nonsyngeneic lymphocytes on differentiation of stem cells in the spleen of irradiated mice

Cell interactions in the immune response in vitro. I. Metabolic activities of T cells in a collaborative antibody response.

AbstractCell collaboration between thymus‐derived (T) and non‐thymus‐derived (B) lymphocytes in induction of the response to SRC was investigated in an in vitro test system. Different populations of T cells were found to vary in helper activity depending on their degree of differentiation following exposure to antigen. Activated thymus cells obtained from the spleen of irradiated mice injected with syngeneic thymocytes and SRC proved the most effective, followed by primed T cells and then unprimed T cells. The interaction between T cells and B cells as measured here was specific for both classes of lymphocytes.The mechanism of T cell action in the collaborative response was analyzed by treating these populations of T cells with agents which became firmly cell bound and could selectively inhibit different aspects of cell metabolism. Mitomycin C was chosen as an inhibitor of DNA synthesis and cell division, actzinomycin D to interfere with RNA synthesis, and antimycin A to block protein synthesis. To exclude significant leakage of inhibitor from T cells to B cells, cultures were stimulated with DNP‐POL, which immunizes B cells directly, as well as with SRC. The development of a normal anti‐DNP response in all cases was confirmation of the integrity of B cell function.Mitomycin treatment (40 μg/ml) of normal T cells or T cells from mice primed to SRC at least four weeks previously markedly reduced helper activity. In contrast, activated T cells which had recently divided in response to antigen and were enriched for specific antigen reactive cells were resistant to mitomycin; thus effective collaboration with B cells could take place in the absence of further differentiation and division by T cells. All T cell populations, whether normal, primed or activated, were sensitive to treatment with actinomycin D at a concentration of 0.1 μg/ml, or 10−5 M antimycin A. Taken together, these experiments suggest that T cells divide and differentiate over a period of approximately 48 h in vitro. During this time antibody production by B cells is initiated by a process requiring active RNA and protein synthesis in T cells.

In vivo requirement for a radiation-resistant cells in the immune response to sheep erythrocytes.

Experiments have been done to establish whether the radiation-resistant or A cell has a specific function in the initiation of an immune response in mice to sheep erythrocytes (SRBC). All previous demonstrations using accessory (A) cells have involved in vitro assays and are possibly explainable as tissue culture artifacts. If A cells are essential, it should be possible to demonstrate their requirement in vivo. Therefore we first established such conditions. Two methods were found for creating an A-cell deficiency in vivo: (a) A cells disappear gradually from the spleens of irradiated mice, presumably by migration since A-cell function was shown not to be decreased by irradiation. If 3 days elapse between irradiation and transplantation of mixtures of bone marrow and thymus cells (which provide B and T but few A cells), the usual synergistic response does not occur. Addition of large numbers of freshly irradiated spleen cells to the mixture of bone marrow and thymus completely restores the immune response. (b) Injection of 10(10) horse erythrocytes into mice suppresses A-cell activity in these mice 24 hr later; a much reduced response to SRBC is obtained when they are given at this time. The response can be partially restored if irradiated spleen cells are given with the SRBC. This observation formed the basis for a quantitative in vivo assay for A cells in which the magnitude of restoration by various suspensions of irradiated cells was used to estimate the A-cell activity of that suspension. A quantitative in vitro assay for A cells was also developed. It was essential for this assay that the total cell number, B-cell number, and T-cell number be kept constant and that only the number of A cells be allowed to vary. Only under these conditions was the response a linear function of the number of A cells added. If the in vivo and in vitro assays are detecting the same class of radiation-resistant cells, the physical properties of the cells active in each assay should be identical. Spleen cells were separated on the basis of both density and sedimentation velocity. Fractions from both separation methods were tested for their content of A cells using both the in vivo and in vitro assays. The density and sedimentation profiles of A cells were similar in both assays. The demonstration that a radiation-resistant cell is required in vivo and that this cell has properties identical to the radiation-resistant cell required in vitro indicates that this cell (the A cell) is directly involved in the initiation of an immune response to erythrocyte antigens.

Migration of transplanted hematopoietic stem cells to the spleen of irradiated mice.

Migration of transplanted hematopoietic stem cells to the spleen of irradiated mice.

Plaque-Forming Cells of Two Different Origins in Single Hemolytic Foci

Abstract When small numbers of spleen cells and heterologous red blood cells as antigens are injected into lethally irradiated mice, regions of hemolytic antibody-forming cell activity—hemolytic foci—can be detected in the spleens of these recipient mice. It has been shown that the number of hemolytic foci in a recipient spleen is directly proportional to the number of cells injected (1, 2), suggesting that each focus represents a single clone of antibody-forming cells, i.e., plaque-forming cells arising from one precursor cell only. Recent observations indicate, however, that formation of hemolytic foci may depend on interaction between at least two cell types. Claman et al. (3) showed that the spleens of irradiated mice injected with sheep red blood cells (SRBC) and either bone marrow or thymus cells did not develop hemolytic foci but that foci were seen when mice were injected with both cell types.

Cell to cell interaction in the immune response. II. The source of hemolysin-forming cells in irradiated mice given bone marrow and thymus or thoracic duct lymphocytes.

The number of discrete hemolytic foci and of hemolysin-forming cells arising in the spleens of heavily irradiated mice given sheep erythrocytes and either syngeneic thymus or bone marrow was not significantly greater than that detected in controls given antigen alone. Thoracic duct cells injected with sheep erythrocytes significantly increased the number of hemolytic foci and 10 million cells gave rise to over 1000 hemolysin-forming cells per spleen. A synergistic effect was observed when syngeneic thoracic duct cells were mixed with syngeneic marrow cells: the number of hemolysin-forming cells produced in this case was far greater than could be accounted for by summating the activities of either cell population given alone. The number of hemolytic foci produced by the mixed population was not however greater than that produced by an equivalent number of thoracic duct cells given without bone marrow. Thymus cells given together with syngeneic bone marrow enabled irradiated mice to produce hemolysin-forming cells but were much less effective than the same number of thoracic duct cells. Likewise syngeneic thymus cells were not as effective as thoracic duct cells in enabling thymectomized irradiated bone marrow-protected hosts to produce hemolysin-forming cells in response to sheep erythrocytes. Irradiated recipients of semiallogeneic thoracic duct cells produced hemolysin-forming cells of donor-type as shown by the use of anti-H2 sera. The identity of the hemolysin-forming cells in the spleens of irradiated mice receiving a mixed inoculum of semiallogeneic thoracic duct cells and syngeneic marrow was not determined because no synergistic effect was obtained in these recipients in contrast to the results in the syngeneic situation. Thymectomized irradiated mice protected with bone marrow for a period of 2 wk and injected with semiallogeneic thoracic duct cells together with sheep erythrocytes did however produce a far greater number of hemolysin-forming cells than irradiated mice receiving the same number of thoracic duct cells without bone marrow. Anti-H2 sera revealed that the antibody-forming cells arising in the spleens of these thymectomized irradiated hosts were derived, not from the injected thoracic duct cells, but from bone marrow. It is concluded that thoracic duct lymph contains a mixture of cell types: some are hemolysin-forming cell precursors and others are antigen-reactive cells which can interact with antigen and initiate the differentiation of hemolysin-forming cell precursors to antibody-forming cells. Bone marrow contains only precursors of hemolysin-forming cells and thymus contains only antigen-reactive cells but in a proportion that is far less than in thoracic duct lymph.

Circadian Rhythmicity in the Effect of Irradiation on the Endogenous Colony Formation in the Spleen of Irradiated Mice

SummaryMice, adapted to cyclic change of light and darkness (12 hours light, 12 hours darkness) were irradiated at different times within the 24 hours. It was observed that after irradiation during the day (14 hours) the number of endogenous colonies in the spleen was higher than that after irradiation at night (20 hours). The lowest number of colonies in the spleen was observed following irradiation 1 hour after the change of light cycle (8, 20 hours). The dose-survival curves for colonies had higher numbers of colonies at 14 hours than that at 8 hours, but the D0 was similar at both times. The transplantation of bone-marrow and spleen cells indicated a higher colony forming ability for cells from adapted mice killed in the middle of the day, than from those killed soon after change of light cycle.The changes in rectal temperature and oxygen consumption of adapted mice followed a contrary course as compared with changes in the number of colonies in spleen after irradiation at different times of the day. ...