Albert A. Nordin

Studies of the immunological capacity of germ-free mouse radiation chimeras. I. Chimerism and humoral immune response.

Abstract The genetic origin of both the functional lymphoid cell and progenitor cell populations of germ-free mouse radiation chimeras was assessed by indirect immunofluorescence (IIF), anti-H-2 cytotoxicity, and survival of lethally x-irradiated secondary recipients of chimera cell populations. These studies demonstrated that germ free C3H/He mice given 1000 R and 107 DBA/2 bone marrow cells express H-2 antigens on their lymphoid and bone marrow cell populations characteristic of the DBA/2 donor. These cells persist for at least 14 months postirradiation and bone marrow transplantation. However, these allogeneic mouse radiation chimeras have a reduced humoral immune response to sheep erythrocytes (SRBC). This decreased humoral immune capacity as assessed by kinetic studies of the spleen plaque-forming cell (PFC) response is present throughout the life span of the chimera. The γ1 PFC response shows extreme depression. The reduced humoral immune responsiveness to the thymusdependent SRBC antigen is considered to be due to the absence or malfunctioning of a thymocyte population.

Studies of the immunological capacity of germ-free mouse radiation chimeras. IV. Cell-mediated immunity.

Abstract The cell-mediated immune (CMI) response of germ-free mouse radiation chimeras was compared with that of conventional mice. Spleen or thymus cells from chimeric or normal mice were injected intravenously into lethally irradiated, allogeneic hosts. Spleens of the irradiated hosts were assayed for effector cells using the 51 Cr release assay. Spleen cells from syngeneic and allogeneic chimeras and normal mice were equally active in giving rise to effector cells. However, thymus cells from allogeneic chimeras were completely inactive within 9 months post-bone marrow transplant while thymus cells from syngeneic chimeras and normal mice still remained functional. Although allogeneic chimeras contain cells potentially reactive toward host antigens, cells cytotoxic to host antigens were not detectable. In addition, these studies indicate the helper cell and effector cell, both associated with T-derived lymphocytes, represent two different populations.

Modulation of natural cytotoxicity by alloantibodies: IV. A comparative study of the activation of mouse spleen cell cytotoxicity by anti H-2 antisera, interferon, and mitogens

Comparisons were made between the characteristics of cytotoxicity activation in mouse spleen cells, induced by specific anti-H-2 antiserum, interferon (IF), interferon inducer (poly (I:C)), and mitogens (concanavalin A (Con A), pokeweed mitogen (PWM)). Important differences were found in the cytotoxicity activation associated with these agents: (a) The cytotoxic enhancing effect of anti-H-2 antiserum was comparatively rapid and was more pronounced in the first 4 hr of the assay, whereas, IF, poly (I:C), and the mitogens were most effective at 20 hr. (b) Anti-IF antiserum could neutrilize the stimulatory effects of IF and poly (I:C) on the cytotoxic activity of mouse spleen cells but had no influence on the stimulatory effects of anti-H-2 antiserum, PWM, and succinyl Con A. (c) The stimulatory effect of anti-H-2 antiserum was target specific and was observed when the K562 cell line was used as the target but not when YAC, P815, or WFu/G1 target cell lines were used. Stimulatory effects of all the other agents were nonspecific and did not depend on the target cells employed. (c) Cellular fractionation studies suggested that the alloantisera and interferon directly activated the natural killer (NK) cells without any participation of T-cells, B-cells, or macrophages. Both T-cells and NK cells, however, contributed to the mitogen-enhanced cytotoxicity of mouse spleen cells. These results indicated that the mechanism by which anti-H-2 antisera induce NK augmentation in mouse spleen cells is distinct from that of interferon and mitogens.

Studies of the immunological capacity of germ-free mouse radiation chimeras: II. Thymus cell function in a humoral immune response to sheep erythrocytes

Abstract Experiments described here were undertaken to determine the reason for the depressed humoral immune response in germ-free mouse allogeneic radiation chimeras. Indirect immunofluorescence using the theta (θ) antigen as a marker demonstrated that about 10% of the nucleated cells in the spleen of both allogeneic and syngeneic chimeras bear the θ antigen. One type of in vivo cell transfer assay employed to determine the capacity for “helper” function of thymocytes revealed that allogeneic chimera thymocytes were only 7–18% as efficient in “helper” function as normal thymocytes. A second type of in vivo cell transfer assay demonstrated that the presence of intact normal thymic stroma had no effect on the “helper” inefficiency of thymocytes obtained from allogeneic radiation chimeras.

The Occurrence of Plaque Forming Cells in Normal and Immunized Conventional and Germfree Mice

Summary The level of normal PFC to SRC and the immune response to 4 × 108 SRC was determined in germfree mice and compared to that of CFW conventional mice. All three strains of germfree mice had normal PFC in the spleen but the CFW mice had fewer than either the Balb/c or ICR mice. The normal PFC in germfree CFW mice was approximately one half the level determined in conventional CFW mice. There was however no correlation between the level of normal PFC and the immune response to SRC. The direct and indirect technique for detecting cells producing antibody to SRC demonstrated no difference in the immune response of germfree and conventional mice. The number of PFC detected by the direct technique on any day after the injection of SRC was slightly lower in the germfree mice but otherwise the kinetics of the immune response were the same as those observed in conventional mice. The indirect technique demonstrates that the PFC synthesizing antibody other than IgM may occur sooner in germfree but otherwise is similar to that of the conventional mice. Antibody titers in the serum of CFW mice were determined with and without treatment with BME and the titers agree with the results of the PFC assays.

The effect of cytosine arabinoside upon the primary immune response in vitro.

Summary The effect of cytosine arabinoside, an inhibitor of DNA synthesis, upon the primary induction of plaque-forming, anti-body-producing cells to sheep erythrocytes was investigated using mouse spleen fragments in vitro. It was found: (i) During the first 12 hr, following antigenic stimulation, the immune response proceeded undisturbed by the presence of the drug, (ii) If the inhibitor was permitted to act for the first 18 hr, the number of plaque-forming cells was reduced to 63% of control values. (iii) Extension of the period of action of the drug to the first 24 hr further reduced the number of plaque-forming cells to 33%, and if the inhibitor was allowed to act for the first 48 hr, total suppression of the immune response was observed. These findings indicate that DNA synthesis, essential for the undisturbed progression of the primary immune response as observed in the system employed, is initiated about the eighteenth hour after antigenic stimulation.

Primary Induction of Plaque Forming Antibody Producing Cells in Spleen Organ Culture

Summary Primary stimulation with sheep erythrocytes of mouse spleen fragments in vitro induced plaque forming antibody producing cells. All spleens tested gave fragments that responded. The percentage of responding fragments from individual spleens varied from 47 to 87%. On the fifth day after stimulation, the numbers of plaque forming cells in stimulated cultures averaged 112 per 106 cells with a range from < 1 to 1080 per 106 cells. Values significantly above the controls were reached, on this day in 72% of the cultures. In addition to 19S antibody producers, evidence of induction of cells making other types of immune globulins was obtained. Free antibody was detected as agglutinins in the (tissue culture medium.

Cellular requirements for the expression of IgM immunological memory in vitro

Abstract In vitro culture techniques have been used to compare the direct (IgM) plaqueforming cell (PFC) response to heterologous erythrocytes (RBC) by normal mouse spleen cells and spleen cells from mice injected intravenously with 5 × 10 4 RBC ten days previously [low dose primed (LDP)]. Although LDP mice fail to undergo a significant primary PFC response, their spleen cells are capable of a secondary or enhanced PFC response in vitro . The secondary PFC response is shown to be a function of: (A) an increase in the frequency of immunocompetent cells or units (IU) due to in vivo priming, and (B) an increased number of PFC generated per IU subsequent to in vitro stimulation. The latter increase is shown to be mediated through a shorter PFC doubling-time during logarithmic expansion of the PFC population. Analysis of nonadherent spleen cell dose response experiments indicate that two nonadherent cell types interact in the secondary response. Subsequent cocultivation experiments suggested that both of these cell types must be “primed” to allow induction of a secondary response. Although adherent cells are required for the secondary response, normal splenic adherent cells serve as equivalent substitutes for LDP adherent cells.