Prostaglandin synthesis in spleen cell cultures of mice injected with Corynebacterium parvum.

Spleen cells of C57BL/6 mice produced high amounts of PGE in vitro when tested 5 to 10 days after injection of heat-killed C. parvum organisms. Little or no PGE was produced by spleen cells from untreated mice or from mice injected with a strain of coryneform bacteria that does not stimulate the lymphoreticular system of mice. Significant release of PGE from spleen cells of C. parvum injected mice could be detected as early as 30 min after initiating the cultures and maximal levels were usually seen after 48 hr. Treatment by indomethacin completely abolished this PGE production. Removal of the adherent population from the spleen cell suspension resulted in markedly decreased levels of PGE, but PGE release of the remaining population was never completely abolished. These data suggest that the cells responsible for most of the PGE synthesis in this system were adherent cells, presumably macrophages. The levels of PGE produced in spleen cells of C. parvum-treated mice were further increased by in vitro addition of C. parvum. This effect could also be observed after addition of zymosan particles indicating that it was not an immunologically specific effect. The reported data suggest that prostaglandins may represent important mediator molecules of the described immunostimulatory and immunosuppressive effects of C. parvum.     

Stimulation of “nude” spleen cells in vitro under the influence of thymus lymphocytes

Spleen cells from “nude” mice (nu/nu) were stimulated in vitro with sheep erythrocytes. After 4 days of in vitro incubation only a few plaque-forming cells were found in these cultures. This immunological deficit can be restored by adding thymus lymphocytes from allogeneic donors to nu/nu spleen cultures. The restoration is not successful when thymocytes are prevented from proliferating, by irradiation or with Mitomycin C, or when their DNA dependent RNA synthesis is suppressed by actinomycin D.     

Alternative induction of alpha/beta interferons and gamma interferon by listeria monocytogenes in mouse spleen cell cultures

Production of interferon (IFN) by Listeria monocytogenes (LM) in nonimmunized mouse spleen cell cultures was studied. IFN-gamma defined by virtue of its acid stability and antigenicity was produced in spleen cell cultures obtained from ddY mice, C57BL/6 mice, and BALB/c mice in response to heat-killed (HK) LM within 24 hr. On the other hand, production of IFN-alpha/beta was demonstrated in spleen cell cultures obtained from one of four nude mice (BALB/c, nu/nu). Therefore, it is important to know the reason why the spleen cells of mice other than nude mice did produce only IFN-gamma, but did not produce IFN-alpha/beta in response to HK-LM. Spleen cells obtained from ddY mice were fractionated, and the cellular source for IFN production of either IFN-alpha/beta or IFN-gamma induced by HK-LM was investigated. IFN-gamma was produced only by a mixture of T lymphocytes (nylon wool-nonadherent, Thy-1-positive cells) and macrophages by HK-LM. Neither T lymphocytes nor macrophages alone produced IFN by HK-LM. Macrophage-depleted spleen cells produced neither IFN-gamma nor IFN-alpha/beta, but these cells acquired the ability to produce IFN-alpha/beta, not IFN-gamma, only when they had been treated with IFN-alpha/beta. A possible mechanism of both IFN-gamma and IFN-alpha/beta induction by Listeria in mouse spleen cell cultures is discussed.     

Mechanism for Macrophage Activation against Corynebacterium parvum—Participation of T Cells and Its Lymphokines

It is well known that Corynebacterium parvum activates macrophages to produce tumor necrosis factor (TNF). It is suspected that the activation of macrophages by C. parvum requires T-cell participation. The purpose of this study was to confirm that T cells participate in the activation of macrophages by C. parvum. TNF production in vitro from the spleen cells of BALB/c- + / + mice was abrogated completely by the pre-treatment of spleen cells with anti-Ia antiserum and complement, indicating that Ia+ cells are the source of TNF. TNF production was not elicited at all in BALB/c-nu/nu mice. However, there was an increase in the number of Ia+ cells as well as an increase in the weight of spleen and liver. Supernatant from a culture of spleen cells stimulated with phytohemagglutinin-P (a PHA-induced lymphokine) made it possible for BALB/c-nu/nu mice to produce TNF, associated with an induction of Lyt-1⁺ cells and Lyt-2⁺ cells. However, treatment with the lymphokine did not augment the increases of Ia⁺ cells or liver and spleen weights. These results suggest that increasing the number of Ia⁺ cells is not sufficient to bring about TNF production; Ia⁺ cells must also be stimulated by T cells or T-cell lymphokines in order to produce TNF. These results suggest that T cells play an essential role in the activation of Ia⁺ cells against C. parvum.     

Separation of functionally distinct subpopulations of Corynebacterium parvum-activated macrophages with predominantly stimulatory or suppressive effect on the cell-mediated cytotoxic T cell response.

Peritoneal cells (PEC) from mice injected ip with Corynebacterium parvum (CP) showed greatly enhanced suppressive activity on the growth of syngeneic tumor cells and on the generation of alloreactive cytotoxic T lymphocytes (CTL) in vitro. On the other hand, CP-activated PEC exhibited increased immunostimulatory (accessory or A cell) activity as measured by the restoration of the CTL response of nonadherent spleen cells. After fractionation of the CP-activated PEC according to cell size by velocity sedimentation, the mutually antagonistic A cell and immunosuppressive activities were clearly separated and found to be associated with functionally distinct subpopulations of macrophages. Thus A cell function was detected in fractions rich in small and medium sized macrophages which were probably derived from recently arrived monocytes. Immunosuppressive (and anti-tumor) activity was associated with the largest macrophages which were almost devoid of A cell function and probably represented a highly activated and differentiated macrophage subpopulation.     

Spontaneous rosette formation of rat thymus cells with guinea pig erythrocytes.

C57 black mice were immunised intraperitoneally with a DBA2 lymphoma (SL2). Fourteen days later spleen cells were prepared. These cells lysed the specific target (SL2) in vitro. Spleen cells were cultured for 24 hr at 37 ° C. Cell-free culture supernatants contained IgG and lysed SL2 cells either in the presence of a source of complement or in the presence of a monolayer of macrophages (a good source of antibody-dependent effectors). The cells producing cell-dependent antibody adhered to nylon wool and were unaffected by anti-theta serum. It was found that the production of antibody in vitro did not make a significant contribution to the observed cytolysis of SL2 by sensitised spleen cells. This effect was mediated by thymus-derived lymphocytes.     

Transplantation antigen expression on murine trophoblast detection by induction of specific alloimmunity

Cell-mediated immune responses to murine embryonic trophoblast cells were investigated using lymphocyte trophoblast cultures (LTC) and cell-mediated lympholysis (CML). Spleen cells from CBA (H-2^k) or C57BL/6 (H-2^b) mice hyperimmunized with 3.5-day-old Balb/c (H-2^d) blastocysts did not undergo DNA synthesis after in vitro exposure to Balb/c blastocyst outgrowths nor were cytotoxic lymphocytes (CTL) generated against H-2^d alloantigens. Splenocytes from Balb/c mice presensitized with semiallogeneic (Balb/c female × C57BL/6 male) trophoblast cells derived from 17- to 20-day placental tissue expressed a weak proliferative response in the presence of semiallogeneic placental trophoblast and produced a moderate number of CTL against H-2^b (paternal strain) alloantigens when compared to mixed lymphocyte cultures (MLC) between Balb/c responder and semiallogeneic (stimulator) spleen cells. CTL were also generated in vitro after splenocytes from Balb/c mice hyperimmunized with semiallogeneic spleen cells were restimulated in vitro with placental trophoblast cells. These studies showing that early-stage trophoblast cells fail to evoke transplantation immunity and placental trophoblast is capable of generating alloimmunity only after combined in vivo hyperimmunization with in vitro restimulation suggest that these trophoblast cells are poorly immunogenic due in part to the relatively weak functional expression of major transplantation antigens.     

Production of an antiviral factor by murine spleen cells after treatment with Corynebacterium parvum.

We have previously shown that injection of Corynebacterium parvum (CP) in mice protected them against lethal encephalitis induced by herpes simplex virus, (HSV). It is shown here that spleen cells of CP-injected mice in vitro produce a factor capable of inhibiting the replication of HSV in mouse embryo fibroblasts (MEF). A similar activity was produced after in vitro exposure of spleen cells from untreated mice to CP. CP was only slightly mitogenic in contrast to phytohemagglutinin (PHA), concanavalin A (Con A), and bacterial lipopolysaccharide (LPS) which were strongly mitogenic but did not induce antiviral activity high enough to be detected in the HSV-MEF system. The activity produced by CP-treated spleen cells appeared to be interferon since it was trypsin sensitive and species specific and not virus specific, and since preincubation of the cells was required to demonstrate an antiviral effect. However, the identity of CP-induced interferon with any of the previously described subclasses of interferon remains to be determined.     

Activation and mechanism of action of suppressor macrophages

Intravenous administration of Corynebacterium parvum to alloimmunized mice activates splenic suppressor macrophages that effectively curtail primary and secondary generation of cytotoxic T lymphocytes (CTLs) in vitro. CTL generation was significantly inhibited in suppressed primary cultures by Day 3, the earliest time point that activity is first detected in control cultures. Suppressor macrophages had to be present during the first 24–48 hr of culture to effectively curtail the generation of CTLs. However, if suppressor macrophages were reactivated by 48-hr in vitro culture and then added to primary sensitizations that had been initiated 48 hr previously, they were capable of significant suppression. Suppressor cells produced a soluble factor that mediated the inhibition of CTL generation. The production or action of this factor could not be counteracted by indomethacin.     

Immunosuppressive Activities of Peritoneal and Splenic Macrophages in Murine Leprosy: Effect on Lymphocyte Transformation and Tumor Growth

The ability of peritoneal macrophages (PM) and splenic macrophages (SM) to suppress tumor growth and lymphocyte transformation in vitro was studied in infected mice with Mycobacterium lepraemurium (MLM). Both PM and SM of leprous mice showed cytostatic activity against tumor cells in vitro. However, such cells showed significantly less cytostatic activity on a per cell basis than highly activated macrophages obtained from Corynebaclerium parvum-immunized mice. Furthermore, this cytostatic activity declined as the infection progressed. Mitogen-induced transformation of splenic lymphocytes was also suppressed in the presence of adherent PM and SM from leprous mice. PM from leprous mice showed significantly less activity than PM from C. parvum-immunized mice in terms of suppression of lymphocyte transformation. Moreover, PM from leprous mice treated with C. parvum or sodium thioglycollate broth demonstrated significantly less ability to suppress lymphocyte transformation than did PM from similarly treated normal mice or untreated leprous mice. These findings demonstrated that MLM infection stimulates the mononuclear phagocyte system but does not activated it to the extent that it confers enhanced resistance to MLM on the host.     

Role of cyclic AMP on differentiation of T- and B-lymphocytes during the immune induction.

Spleen cell cultures from genetically thymus-deficient nude mice were restored with a T-cell replacing factor obtained from normal spleen cells of Balb/c-Ig^b mice stimulated with concanavalin A. Treatment of these cultures with an inhibitory dose of cyclic AMP did not result in reduction of the number of specific antibody-forming cells after stimulation by antigen, whereas the same treatment led to inhibition in cultures restored with normal hydrocortisone-resistant thymus lymphocytes. Further experiments lead to the conclusion that the early effect of cAMP on the immune induction seen in vitro reflects inhibition of the production or secretion of a T-cell factor which is a prerequisite for triggering B-cells with a thymus-dependent antigen.     

Interferon inhibition of the primary in vitro antibody response to a thymus-independent antigen

Partially purified and crude mouse L cell interferon preparations inhibited the in vitro plaque-forming cell (PFC) response of mouse C57B1/6 spleen cells to the T-cell independent lipopolysaccharide antigen of Escherichia coli 0127. PFC responses of 5-day cultures were inhibited approximately 70–90% by 100–200 NIH reference units of interferon/culture. A similar inhibitory effect was obtained with spleen cells from athymic (nude) mice homozygous for the nu/nu allele. Spleen cultures depleted of adherent cells were also inhibited in their anti-0127 PFC response by interferon. Interferon, then, appears capable of inhibiting the PFC response to E. coli 0127 via direct action on B cells. Heating experiments along with the use of interferon preparations of different specific activities suggest that the inhibition was due to the interferon in the preparations.     

Biological effects of the adjuvant Corynebacterium parvum. II. Evidence for macrophage-T-cell interaction

The mechanism underlying the markedly reduced PHA responsiveness of spleen and peripheral blood lymphocytes from Corynebacterium parvum-treated mice is due to inhibition of the responsive T-lymphocytes by C. parvum-activated macrophages. Inhibition is a result of a qualitative rather than quantitative change in the macrophage population and GVH-activated macrophages behave similarly. Corynebacterium parvum-activated macrophages need to be viable and will inhibit normal lymphocytes. This inhibitory effect appears to be mediated through cell-cell contact.     

Plaque-forming cells fater in vitro stimulation with theta-AKR antigen

Spleen cells of inbred mice strains carrying θ-C3H allele have been cultured in the presence of AKR thymus cells and their in vitro primary PFC response against thymic alloantigen θ-AKR was studied.The responses of a magnitude which was comparable with that obtained in previous in vivo experiments were obtained 4 days after stimulation. The strain-dependent variability of the magitude of anti-θ-AKR responses was observed in vitro. RR and C58/J spleen cells produced much more PFC than C57BL/6J and DBA/2J spleen cells. This was in agreement with previous in vivo studies on the genetic control of the anti-θ AKR responses.In the absence of AKR thymus cells, spleen cells of high responders, RR, developed in vitro PFC which released antibodies lytic to AKR thymus cells. Their number was ten-times lower than in stimulated cultures. Spleen cells of all strains tested produced also small numbers of PFC secreting antibodies against θ-identical allogenic thymus cells and even to syngenic thymus cells.     

Inhibition of memory cell-mediated cytotoxic response by systemic administration of Corynebacterium parvum.

Intravenous administration of Corynebacterium parvum to mice during a developing immune response to alloantigens resulted in the marked inhibition of the generation and expression of memory cell-mediated cytotoxic response in the spleen. The inhibition was observed following rechallenge in vivo or by in vitro culturing with the same alloantigen. The impairment in vitro was due, in part, to the generation of regulatory cells which were non-T phagocytic cells, probably macrophages activated by C. parvum administration. These suppressor macrophages appear to act by inhibiting proliferation and clonal expansion of memory cytotoxic cells.     

Influenza virus-induced interferon production in mouse spleen cell culture: T cells as the main producer.

Interferon production by spleen cells from unimmunized C3H mice challenged in vitro with influenza virus AO/PR8 was investigated. Glass-nonadherent cells (lymphocytes) produced significant levels of interferon, although cocultivation of glass-adherent macrophages was needed for optimal production. Treatment of the cells with antithymocyte serum and complement markedly reduced the interferon production. When glass-nonadherent cells were fractionated on a nylon wool column, the T-cell-enriched fraction consistently produced more interferon than the B-cell-enriched fraction. It is concluded that T cells are an important producer of interferon in spleen cell cultures from normal mice upon challenge with influenza virus, although non-T cells (macrophages and B cells) also may produce interferon under suitable conditions.     

Mechanisms of antibody formation: I. Early in vivo proliferation of mouse spleen T cells as an initial step in antibody formation

Spleen cultures prepared from mice injected 24 hr earlier with 2 × 10⁶?2 × 10⁸ sRBC and challenged in vitro with sRBC produced 10 times more anti-sRBC IgM PFC than cultures prepared from uninjected mice. The effect was specific for the particular species of foreign RBC injected in vivo. In vitro responses to TNP were also increased in spleen cultures prepared from animals injected 24 or 12 hr earlier with carrier RBC alone, directly implicating carrier-specific T cells in this process. Similar enhancements of PFC formation occurred in cultures prepared from mice which had been injected with sRBC 24 and 48 hr earlier, but which were exposed to lethal irradiation at 1 hr after injection of antigen, if their spleens were shielded extracorporeally during irradiation. This finding indicated that in vivo recruitment of antigen-reactive extrasplenic X-ray-sensitive cells from the circulating lymphocyte pool by the spleen could not account for the observed enhancement.Proliferation in the spleen of antigen-reactive T cells, commencing 12–20 hr after the administration of antigen, was demonstrated by the tritiated thymidine pulse technique. An 8-hr hot-pulse given to spleen cell cultures from normal animals at 20 hr after in vitro challenge with antigen did not affect the rate of generation of IgM-producing cells; however, administration of a similar pulse to cultures which were initiated at 12 or at 20 hr after the in vivo injection of sRBC eliminated the enhanced generation of PFC and delayed the in vitro response to sRBC by 24 hr.Spleen cell cultures were prepared from mice which had been injected in vivo with sRBC at 12, 20, and 70 hr earlier, and 8- to 10-hr hot pulses were given immediately after initiation of the cultures. The cultures were then challenged with sRBC-TNP; antibody responses to TNP were greatly reduced in hot-pulsed cultures prepared from mice injected in vivo with carrier RBC at 12 or 20 hr prior to initiation of the cultures. In contrast, antibody responses to TNP observed in hot-pulsed cultures prepared from mice which had been injected with carrier RBC at 70 hr prior to initiation of the cultures were generally similar to those of nonpulsed 70 hr control cultures. This result suggests that the onset of T helper cell proliferation begins within 12–20 hr after injection of antigen, but subsides in vivo within 70 hr. By that time, the antigen-reactive T cells have already differentiated to perform their helper function.In spite of the triggering of T-cell proliferation during the first 24 hr after injection of antigen, spleen cell cultures prepared from mice which had been injected 24 hr earlier in vivo with 2 × 10⁸ sRBC produced only minimal numbers of anti-sRBC PFC if no antigen was added to the cultures. The presence of unprocessed antigen thus appears to be a requirement for B-cell proliferation in vitro, even after T-cell division has been triggered. This finding is consistent with earlier suggestions that the function of “helper” T cells may not be limited to passive transport of antigenic determinants to B cells. Evidence is also presented to support the contention that the antigen-reactive T cell involved in this process may have to undergo cell division in order to develop “helper” capacity.     

Suppressor T cells and suppressor macrophages induced by Corynebacterium parvum

Corynebacterium parvum, injected intravenously into C57B1/6 mice (H-2^b) previously alloimmunized with P815 (H-2^d) mastocytoma cells, generated splenic suppressor cells that inhibited the development of primary cytotoxic lymphocytes in vitro. These suppressor cells differed from those generated by intravenous C. parvum injection of naive C57B1/6 mice. The former suppressor cells were effectively induced by administration of 700 μg of C. parvum whereas the latter suppressor cells were dependent upon higher doses (1400 μg) of adjuvant for their activation. Furthermore, suppressor cells generated in alloimmunized mice could only suppress C57B1/6 anti-P815 in vitro cytotoxic responses whereas suppressor cells generated in naive mice could suppress C57B1/6 anti-CBA (H-2^k) responses as well. Suppressor cells were not H-2 restricted in their action. Fractionation of spleens from alloimmunized, C. parvum-treated mice revealed the presence of suppressor T cells and suppressor macrophages. We were unable, however, to determine which cell was responsible for “antigen specificity” of suppression since the fractionation procedures seemed to trigger both suppressor cell types prior to adding them to the primary culture.     

Immunosuppressive activity of murine newborn spleen cells. I. Selective inhibition of in vitro lymphocyte activation.

Cell-mediated immune responses to newborn lymphocyte alloantigens were investiated using mitogen activation, mixed lymphocyte reaction (MLR) and cell-mediated lympholysis (CML). Spleen cells from 1- to 5-day-old (C57BL/6 × Balb/c) F₁ mice co-cultured with maternal strain (BALB/c) splenocytes did not affect DNA synthesis of maternal strain cells in the presence of concanavalin A or phytohemagglutinin. Newborn cells did inhibit the lipopolysaccharide response of maternal strain lymphocytes and these cells also depressed DNA synthesis when added to MLR cultures of BALB/c and C57BL/6 spleen cells. Newborn cells expressed poor stimulatory capacity in semiallogeneic MLR and also caused marked inhibition of DNA synthesis when added to semiallogeneic MLR containing BALB/c (responder) and CB6F₁ adult splenocytes (stimulator). The suppression of MLR by neonatal cells persisted for the first 2 weeks of life and was associated with a soluble factor released during culture. The suppressive activity was almost completely abrogated after depleting the T-cells from newborn splenocytes. However, these same cells did not interfere with the in vitro generation of cytotoxic lymphocytes in the CML assay. The selective immunosuppressive properties of newborn spleen cells may be important during pregancy by protecting the immunologically alien fetus from rejection by the mother.     

Trypanosoma cruzi: In vivo and in vitro correlation between T-cell activation and susceptibility in inbred strains of mice

The in vivo susceptibility of several inbred strains of mice to the Y and CL strains of Trypanosoma cruzi was compared to the in vitro ability of spleen cells from infected mice to generate factor(s) able to activate macrophages to a trypanocidal state. Spleen cells from resistant immune mice generate higher levels of the factor(s) and do so at earlier times during infection than those of susceptible mice. The spleen cells capable of generating the in vitro factor(s) are also capable of conferring resistance upon passive transfer. Removal of immunoglobulin-bearing cells from the immune spleen cell population did not affect either transfer of protection in vivo or generation of the factor(s) in vitro. The cellular basis underlying the differences between susceptible and resistant mouse strains has not yet been determined.