Cell-impermeable Diffusion Chambers Research Articles

CD4+ T cell mediated destruction of xenografts within cell-impermeable membranes in the absence of CD8+ T cells and B cells.

Xenogeneic cells encapsulated in cell-impermeable diffusion chambers die within 3 weeks when implanted into immunocompetent animals but not when implanted into immunodeficient animals. To determine which cells are necessary for this observation, we depleted normal mice in vivo of either CD4+ or CD8+ T cells using monoclonal antibodies. We also reconstituted the immune system of athymic CBA mice (T-lymphocyte deficient) and C.B17 SCID mice (T- and B-lymphocyte deficient) with different cell subsets from normal CBA and BALB/C mice, respectively. Depleted or reconstituted mice were implanted with a diffusion chamber containing COS (monkey kidney) cells. Membrane enclosed xenografts survived in CD4+ T cell depleted mice but not in CD8+ T cell depleted or nondepleted control mice. Encapsulated xenografts survived when implanted into either athymic or SCID mice but were destroyed in reconstituted athymic and SCID mice. Furthermore, encapsulated xenogeneic cells were destroyed in athymic or SCID mice reconstituted with CD4+ cell preparations depleted of CD8+ cells and/or B cells. In contrast, encapsulated xenogeneic cells were not destroyed in athymic or SCID mice reconstituted with CD8+ cell preparations depleted of CD4+ cells. These studies highlight the critical role of CD4+ T cells, in the absence of CD8+ cells and B cells, in the processes leading to the ultimate destruction of encapsulated xenografts. Because of the use of cell-impermeable membranes in these studies, the most likely involvement of CD4+ T cells is in the indirect antigen recognition by these cells and subsequent stimulation of inflammatory cells.

Recruitment of alloreactive natural killer cells to the rat peritoneum by a transfected cell line secreting rat recombinant interleukin-2

In order to obtain large numbers of natural killer (NK) cells from single rats for functional studies, we have devised a method for the generation of IL-2-activated NK cells in vivo. Rats were implanted intraperitoneally with cell-impermeable diffusion chambers (DC) containing cultures of transfected Chinese hamster ovary (CHO) cells secreting rat recombinant interleukin-2 (rIL-2). This resulted in a dramatic increase in the peritoneal exudate cell (PEC) number with a peak (300–1000 × 10 6) 1 week after implantation. The majority were mononuclear cells of which a large proportion were CD3 −NKR-P1 + NK cells, but with substantial numbers of macrophages (Mφ) and CD3 +8 +NKR-P1 + T cells also. The NK activity against standard tumor target cells was high among PEC from six different inbred rat strains tested. However, the NK cell-mediated reactivity against concanavalin A (ConA)-activated T cell blasts from a panel of major histocompatibility complex (MHC) congenic strains differed widely. PEC from some strains (PVG, LOU/C, and AO) efficiently lysed all the MHC-disparate lymphoblasts. In other strains (BN and LEW) more restricted allorecognition repertoires were observed, whereas PEC from one strain (DA) were unresponsive. The secretion of rat rIL-2 intraperitoneally did not lead to a significant increase in the IL-2 level in the blood or in the total number or activity of NK cells in blood and spleen. The present method represents a most potent technique for generating large numbers of functional rat NK cells and shows the high efficiency with which IL-2 can induce NK cell recruitment in vivo.

The proliferation and life-span of rat large granular lymphocytes: effects of cytokines.

Pulse labeling of large granular lymphocytes (LGL) with [3H]thymidine for 1 h in vitro showed that 1%-7% of LGL were in S phase in the blood, spleen and liver of unstimulated euthymic and athymic rats as scored with autoradiography. Repetitive injections of [3H]thymidine over 2-7 days revealed that about half of the blood and spleen LGL had formed by division of precursors during this week. Stimulation of rats with the interferon inducer poly(I).poly(C) increased the proportion of S phase LGL rapidly and simultaneously in the blood, spleen and liver, so that by 20 h after stimulation ca 30% of LGL were in the S phase in these organs. This LGL proliferation was accompanied by an increased number of LGL in all three compartments 48-96 h after poly(I).poly(C) injection. Blood LGL cultured in cell impermeable diffusion chambers in the peritoneal cavity of poly(I).poly(C)-stimulated rats exhibited enhanced natural killer (NK) activity but no proliferative response, indicating that mature LGL were not induced to undergo blastogenesis by poly(I).poly(C) in vivo. Pretreatment of NK cells with rat interferon (IFN-alpha/beta) and with human recombinant interleukin 2 (rIL 2) in vitro, showed that these two cytokines, when combined, had opposing effects on NK activity and proliferation: whereas rIL 2 inhibited the IFN-induced augmentation in NK activity, IFN inhibited the rIL 2-induced LGL proliferation.

The "natural resistance" to bone marrow allografts in normal and athymic nude rats. Rapid cytotoxic reactions both in vivo and in vitro.

To elucidate the rapid destruction of allogeneic bone marrow (BM) grafts in nonsensitized rats we have assessed: (a) the 21-h tissue localization of 51Cr-labeled allogeneic vs. syngeneic BM cells, (b) the ability of allogeneic BM cells to proliferate in the BM or irradiated recipients, (c) the survival of allogeneic vs. syngeneic BM cells in cell-impermeable diffusion chambers implanted into the peritoneal cavity of rats, and (d) the destruction of allogeneic vs. syngeneic labeled BM cells in vitro by effector cells from various lympho-myeloid tissues. Some allogeneic BM cells were destroyed within 24 h after transfer to athymic nude rats, thus demonstrating the thymus independence of the cytotoxicity. Furthermore, allogeneic BM cells also failed to proliferate in the BM of irradiated recipients. However, the survival of allogeneic BM cells was not impaired if they were sheltered from host cells within cell-impermeable diffusion chambers over a culture period of 4 days. But when the allogeneic BM cells were similarly cultured in hosts presensitized against the BM donor, a substantial reduction in BM cell survival was observed. The effector cells of allogeneic BM cytotoxicity (ABC) were present in the spleen because PVG-rnu spleen cells were highly effective in lyzing BM cells from the AO, but not the PVG strain in vitro after only 4 h of culture. Some ABC activity in vitro was also found for peripheral blood lymphocytes and lymph node cells, but not for BM cells themselves. Taken as a whole these data provide firm evidence that the "natural resistance" to BM allografts is basically different from conventional immune responses, and in most, but not all respects resembles natural killer activity.

Postnatal Development of T Cells

Abstract The in vitro migration pattern of neonatal spleen cells was studied in a system of grafts: the newborn spleen was grafted under the renal capsule of adult mice, either normal, thymectomized as adults, or T-deprived animals. The influence of thymic humoral factors on these cells was studied by grafting some of the hosts with thymic tissue in cell-impermeable diffusion chambers. The results showed that the cells of the neonatal spleen contain subpopulations that migrate specifically to either the thymus, the spleen, or the lymph nodes and that these migration patterns are not influenced by thymic humoral function. In T-deprived animals; however, in the absence of any thymic influences, cells that migrated to the thymus, only, were present. Under the influence of thymic humoral factors, this thymus-seeking population became minimal while a new population of spleen- and nodeseeking cells was detectable. The results provide evidence to suggest that there is a sequence of maturation in terms of lymphoid organ homing of precursor cells and that one population may generate the succeeding populations. There is also evidence to suggest that a thymic humoral factor may play a role in these changes in migration patterns without necessarily direct correlation with changes in classic criteria of T cell maturation.

Syngeneic Antitumor and Antiembryo Immunization Affecting Pregnancy in Mice

Groups of virgin BALB/c female were immunized against methylcholanthrene, SV40-induced or spontaneous syngeneic sarcoma cells or against syngeneic mitomycin-C blocked embryonic cells. Females were then mated to syngeneic males and observed for pregnancy rate and size of litters. To mimic the antiembryo immunization occurring during normal pregnancy, other experimental groups were added in which midgestational embryo fragments were kept in cell-impermeable diffusion chambers placed in the peritoneal cavity of virgin femals for 20 days, and removed before mating these females with syngeneic males. In all cases, antitumor and antiembryo immunization significantly reduced the number of successful pregnancies after the 1st mating while the second pregnancy appeared to be unaffected by the treatment. A significant reduction in the mean litter size was found, in mice immunized with embryonic tissues or with the SV40-induced sarcoma but not in those immunized with methylcholanthrene-induced or spontaneous tumors.

In vivo inhibition of antitumor immunity by embryonic antigens.

Adult female BALB/c mice were immunized against the 3-methylcholanthrene–induced fibrosarcoma ST2 by growth and excision, and then injected with either neoplastic or embryonic mitomycin-C-blocked syngeneic cells before receiving a subcutaneous challenge of 105 ST2 cells. Other groups of similarly immunized females were intraperitoneally implanted with cell-impermeable diffusion chambers containing neoplastic or embryonic tissues, or mated to syngeneic males, before being challenged with ST2 cells. The mice immunized and injected with blocked ST2 but not with embryonic or antigenically unrelated neoplastic cells showed a resistance to the growth of ST2 significantly lower than that of immune mice given normal adult cells. This was particularly evident when ST2 blocked cells were given parenterally but was also detectable when ST2 blocked cells were cultured within diffusion chambers kept in tumor–excised mice. The syngeneic pregnancy had no effect on the antitumor immunity. In a subsequent study, BALB/c females were challenged with the same number of ST2 tumor cells as above, but the challenge was performed at days +4, +2, 0, –-2, –-4 from excision, and the blocked neoplastic embryonic or normal adult cells were given starting at the day of challenge. It was found that when the challenge was done 4 and 2 days after the excision, the administration of either antigenically-related tumor cells or embryonic cells could reduce the antitumor immune protection, while no such an effect was detectable in the other experimental groups. Thus, in addition to tumor-associated transplantation antigens, embryonic antigens also seem to be able to impair the developing antitumor immunity provided they are given within four days from the excision of the immunizing growth.

Nonimmunogenic sarcomas induced by 3-methylcholanthrene treatment of murine fibroblasts in diffusion chambers.

A 30-mug dose of 3-methylcholanthrene (MCA) was applied for 1 week to normal BALB/c fibroblasts in cell-impermeable diffusion chambers (DC) in the peritoneal cavities of BALB/c micemtwo groups of DC cultures, in which the carcinogen was given during weeks 1 and 5 of cultivation, respectively, were compared for the frequency of malignant transformation and for the immunogenicity of the resulting neoplasms. The cells from each DC were transplanted sc into immunodepressed semisyngeneic mice for assay of their tumorigenicity. Although tumor yield was similar in the 2 groups (25 and 22%, respectively), there was clear difference in immunogenicity; 10 of 16 sarcomas from fibroblasts treated during week 1 of culture were nonimmunogenic, whereas 8 of 9 tumors from the older cultures were immunogenic (P less than 0.02). A kinetic study of normal fibroblasts in DC revealed that cells proliferated rapidly, with a peak at day 4 after seeding, then grew progressively more slowly and ceased to replicate between 14 and 28 days of culture. Thus there was a notable difference at the moment of MCA application in the growth phase of the target cell population of the first as compared with the fifth week of culture, possible related to the different expression of tumor-associated transplantation antigens in the resulting neoplasms.

Kinetics of murine haemopoietic cell proliferation in diffusion chambers.

ABSTRACTMurine bone marrow cells were cultured in cell impermeable diffusion chambers in the abdominal cavities of mice. The kinetics of granulocyte and macrophage formation were studied by stathmokinetic and autoradiographic techniques. During the period of most rapid growth of proliferative granulocytes, their generation time and its different phases were: tc∽ 8 hr, tG1∽ 1·5 hr, ts∽ 5·5 hr, tG2∽ 0·7 hr and tM∽ 0·25 hr.The generation time of macrophages and their precursors was approximately 8 hr. Formation of macrophages was significantly reduced when chamber inoculum was increased, as judged by 3H‐TdR labelling index.

Anti-DNP hemolytic plaques by mouse spleen cells in diffusion chambers.

Spleen cells frcan mice immunized with dinitrophenylated keyhole limpet hemocyanin (DNP-KLH) were restimulated with DNP-KLH in vitro, enclosed in cell-impermeable diffusion chambers and implanted in the peritoneal cavity of irradiated recipient mice. At various intervals, the cells were recwered from the chambers and DKP-specific plaque forming cells (PFC) were enumerated. The numbers of nucleated cells in the chambers declined rapidly during the first day after implantstion then increased between days 3 and 6 and remained at between 30 and 40% of the initial number from day 6 to day 10. Doses of DNF-KLH of 5 to 500 ng induced maximal numbers of PFC, while fewer PEC were obtained Kith lower and higher doses. The numbers of PFC/106 cells declined during the first 2 days of culture in diffusion chambers, then increased explosively between days 3 and 4 and reached a peak on day 5 through 8. At peak response, 5% of the nucleated cells were PFC and in one case the proportion of PFC was 12%. Varying the time be...

The Immunosuppressive Effects of Rauscher Leukemia Virus (RLV) upon Spleen Cells Cultured in Cell-Impermeable Diffusion Chambers

Abstract In mouse spleen cell cultures, keyhole limpet hemocyanin (KLH) inhibited the leukemogenic process induced by Rauscher leukemia virus (RLV). Spleen cells obtained from normal or RLV-infected BALB/C mice were cultured with or without KLH in cell-impermeable diffusion chambers that were implanted in isogeneic hosts. The effects of RLV and KLH upon these cells were determined by quantitative analysis of cell morphology and by measurement of in vitro immunoglobulin synthesis. Increases in blast, myeloid, erythroid and monocytoid cells were found in RLV-infected cultures that were not restimulated with KLH. In RLV-infected cultures that were restimulated with KLH, numbers of blasts and hematopoietic cells were within normal range, but there was a marked increase of plasma cells and of in vitro immunoglobulin synthesis. It is postulated that antigenic stimulation by KLH of RLV-infected cultures shifts the differentiation pathways of a pluripotent stem cell from a blast-hematopoietic cell line into a lymphoid-plasma cell line. The finding that antigenic stimulation of infected cell cultures alters the cellular pathway induced by a leukemogenic virus may provide a new approach to the investigation of regulatory mechanisms and control of cell proliferation and differentiation during leukemogenesis.

The Immunosuppressive Effects of Rauscher Leukemia Virus Upon Spleen Cells Cultured in Cell-Impermeable Diffusion Chambers

Abstract Qualitative and quantitative differences were demonstrated in the effects of Rauscher leukemia virus (RLV) upon various expressions of the immune response. BALB/c mouse spleen cells were inoculated with RLV and cultured in cell-impermeable diffusion chambers. The suppressive effects of RLV on the cellular expression of IgM and IgG memory and on the cell responses to phytohemagglutinin (PHA) and alloantigens were investigated. RLV inoculated at time zero of culture inhibited IgG but not IgM memory. Both responses were depressed when the interval between RLV inoculation and antigenic restimulation was increased. Removal of the macrophage-rich, adherent cell population depressed the IgM but not the IgG response, indicating that the early selective inhibition of IgG memory was not dependent on infection of macrophages by RLV. Incorporation of 3H-TdR was higher in cultures of spleen cells infected with RLV than in non-infected controls. This high background precluded accurate measurement of stimulation of DNA synthesis by RLV-infected BALB/c cells cultured with mitomycin-treated C57BL mouse spleen cells. However, infected cultures responded to PHA stimulation with 3H-TdR incorporation similar to that of the non-infected, PHA-stimulated cells. These data indicated that RLV-induced immunosuppression is not an all-or-none phenomenon; the degree of inhibition appeared to depend on the different immunocompetent units participating in various immune responses.

Staircase Rise in the Antibody-Forming Cell Population in Secondary Response

Abstract Secondary anti-sheep RBC response by dispersed mouse spleen cells cultured in cell-impermeable diffusion chambers was assessed by the hemolytic plaque assay in combination with autoradiography and treatment with vinblastine. The results showed that, as in the spleen of mice undergoing a primary response, growth of the antibody-forming cell population occurred non-randomly in a staircase manner and recruitment of precursor cells into the antibody-forming cell population occurred repeatedly in a non-random manner. However, the size of the recruitment was significantly larger than that in the spleen of mice undergoing a primary response (i.e., a maximum of 40-fold vs 4-fold). Further studies on these precursor cells indicated that they transform into antibody-forming cells after a short but distinct lag following completion of mitosis. Another important observation was that the antigen-triggered proliferation of immunocompetent precursor cells could have occurred as early as 30 min after exposure of dispersed spleen cells to the test antigen, in contrast to a lag of more than 10 hr in the spleen of mice undergoing a primary response.

The Suppressive Effect of Rauscher Leukemia Virus on the Secondary Antibody Response of Spleen Cells Cultured in Cell-Impermeable Diffusion Chambers

Abstract The effects of Rauscher leukemia virus (RLV) on the immune response to sheep red blood cells (SRBC) were studied in BALB/c mice and in isogeneic spleen cells cultured in cell-impermeable diffusion chambers implanted into irradiated hosts. Primary antibody formation to SRBC was depressed in mice inoculated with RLV. Mice injected with SRBC from 0 to 48 hr after virus inoculation had normal 19 S and depressed 7 S antibody production. Both 7 S and 19 S antibodies were depressed when SRBC were inoculated 3 to 15 days after RLV infection. There was a positive correlation between erythroblastic reaction and immunosuppression in two strains of mice: BALB/c was sensitive and C57BL partially resistant to the erythroblastic and immunosuppressive effects of RLV. Antibody production was not impaired in mice inoculated with RLV mixed with anti-RLV mouse antisera providing evidence for the viral specificity of immunosuppression. Antibody production and plasmacytosis were demonstrated in SRBC-primed spleen cells cultured with SRBC in cell-impermeable diffusion chambers implanted into irradiated isogeneic hosts. RLV inhibited the secondary antibody response to SRBC in intact mice and in spleen cells cultured in diffusion chambers. Increase in blast cells, reduction in plasma cells, and depression of antibody formation were found both in primed spleen cells infected with RLV prior to culture and in primed spleen cells simultaneously exposed to RLV and SRBC in diffusion chamber cultures. This system provides a quantitative approach to the study of mechanisms of oncogenic virus-induced immunosuppression at cellular and subcellular levels.

In vitro Antibody Synthesis by Diffusion Chamber Cultures of Spleen Cells

Summary Techniques for the study of antibody production by primed antigen-stimulated spleen cells in cell-impermeable diffusion chambers have been developed. The chambers were recovered from their host animals and incubated in vitro with radioactive amino acids. Antibody production was measured by determining the amount of radioactivity which would specifically bind to erythrocyte stroma of the type used for immunization. In addition, the effect of kiloroentgen doses of radiation on antibody production by plateau phase cultures was studied in a system in which primed spleen cells in cell-impermeable diffusion chambers were irradiated 6 days after antigen stimulation. One and three days later the chamber cultures were incubated in vitro in the presence of 14C-labeled amino acids. The incorporation of radioactivity into total extracellular proteins, γG-globulin, and specific antibody was measured. Isotope incorporation in cultures which had received 10,000 r of x-radiation was qualitatively and quantitatively similar to that by unirradiated controls—as judged by a variety of criteria. It has been shown previously that irradiation of diffusion chamber cultures of antigen-stimulated spleen cells with kiloroentgen doses of x-radiation in the plateau phase of their response leads to a purification of mature antibody-producing cells and virtually eliminates cell multiplication. It is shown here that the surviving immunocompetent cells are as efficient as unirradiated cells in synthesizing antibodies. Thus, it appears that heavily irradiated cultures of lymphoid cells should be useful for further biochemical studies of antibody synthesis and regulation.

Thymic control of cellular differentiation in the immunological system

SummaryThymectomized and intact (non-thymectomized) adult mice were heavily irradiated and given isogeneic bone marrow. In contrast to the intact mice, thymectomized mice failed to recover the capacity to produce normal numbers of plaque-forming cells after antigenic challenge. However, when cell-impermeable diffusion chambers containing thymus were implanted into thymectomized mice, these animals produced significantly larger numbers of plaque-forming cells than did control mice bearing empty diffusion chambers or chambers containing lymph node or spleen. Since the thymus does not influence the differentiation of antigen-sensitive cells into plaque-forming cells, these results indicate that the influence of the thymus is directed at the differentiation of antigen-insensitive precursors in bone marrow to antigen-sensitive cells. The capacity of thymectomized mice bearing thymus-filled diffusion chambers to produce plaque-forming cells indicates that the thymic influence is mediated by a humoral factor.

Thymic control of cellular differentiation in the immunological system.

SummaryThymectomized and intact (non-thymectomized) adult mice were heavily irradiated and given isogeneic bone marrow. In contrast to the intact mice, thymectomized mice failed to recover the capacity to produce normal numbers of plaque-forming cells after antigenic challenge. However, when cell-impermeable diffusion chambers containing thymus were implanted into thymectomized mice, these animals produced significantly larger numbers of plaque-forming cells than did control mice bearing empty diffusion chambers or chambers containing lymph node or spleen. Since the thymus does not influence the differentiation of antigen-sensitive cells into plaque-forming cells, these results indicate that the influence of the thymus is directed at the differentiation of antigen-insensitive precursors in bone marrow to antigen-sensitive cells. The capacity of thymectomized mice bearing thymus-filled diffusion chambers to produce plaque-forming cells indicates that the thymic influence is mediated by a humoral factor.

Influence of bursa implantation upon lymphocytic nodules and plasma cells in spleens of bursectomized chickens.

SummaryImplantation of donor bursa in hormonally bursectomized chickens restored the immunological reactivity by means of a humoral substance capable of passing through cell-impermeable diffusion chambers. Lym-phocytic nodules, shown to develop under the influence of the humoral agent from the bursa, are felt, in part, to be the site of antibody production. Plasma cell development does not appear to be by means of the humoral substance alone.

The Suppressive Effect of Supraoptimum Doses of Antigen of the Secondary Antibody-Forming Response of Spleen Cells Cultured in Cell-Impermeable Diffusion Chambers

Summary The effect of antigen dose on adequately primed spleen cells was studied in cell-impermeable diffusion chambers. Maximum secondary antibody response against sheep red blood cells (RBC) was obtained when 1.2 × 107 spleen cells were cultured with 1.2 × 106 sheep RBC, a ratio of sheep RBC to spleen cells of 1:10. Maximum secondary antibody response against soluble bovine serum albumin (BSA) was obtained when 2.4 × 107 spleen cells were cultured with 50 µg of BSA. A suboptimum response was readily obtained not only by decreasing the antigen dose below the optimum but also by increasing it. Pulse-labeling studies with H3-thymidine were carried out during the log phase of response, when the total number of lymphoid cells remained relatively constant. The results revealed that antigenic stimulation can cause a twofold reduction in the t2 and therefore the t1/2 of the lymphoid cells from approximately 1.0 to 0.5 day. Further autoradiographic studies with use of H3-thymidine, H3-uridine and H3-proline revealed that morphologically distinct blast cells, lymphocytes and plasma cells are in general metabolically distinct, and the decrease in the t2 of the lymphoid cell population after antigenic stimulation is due to preferential proliferation of metabolically active blast cells and plasma cells. Furthermore they showed that the change in antibody output as affected by the dose of RBC antigen can be related mainly to the number of metabolically active plasma cells. Finally, immunofluorescent studies showed that when primed spleeen cells were cultured with over 100 times the optimum dose of BSA the number of functional antibody-forming cells decreased markedly 1 week after culture, and, in addition, the efficiency of these functional cells seemed to be reduced to one fourth of the normal. The significance of these results to the cytokinetics of antibody response is discussed.

Differentiation of Lymphocytes Undergoing an Immune Response in Diffusion Chambers

Summary No transformation of lymphocytes into histiocytes and fibroblasts was observed when cell-impermeable diffusion chambers containing rat thoracic duct lymphocytes were implanted intraperitoneally in unirradiated isologous or homologous recipients. When such chambers were implanted into irradiated heterologous recipients (mice), or when chambers containing these cells together with radiation-killed mouse cells were implanted into irradiated isologous recipients, the number of blast and immature cells increased with a T2 of approximately 13 hr between 2 and 6 days after culture. These cells were shown not to be of host origin. Pulse-labeling studies with tritiated thymidine indicated that they were derived mainly from deoxyribonucleic acid-synthesizing lymphocytes. No attempt was made to determine what cell type or types are the antigen-inducible progenitor cells, for previous studies indicate there is only 1 progenitor cell in approximately 106 lymphoid cells.