In vivo generation of suppressor T cells by thymosin in congenitally athymic nude mice

Treatment of nude mice with thymic factors such as thymosin has been mostly ineffective in generating effector T cells. This study examined the effects of treating nude mice with thymosin fraction 5 on the induction of cells that could participate in and/or regulate cytotoxic T lymphocyte (CTL) generation by normal spleen cells in vitro. Splenic lymphocytes from BALB/c nude mice injected with thymosin fraction 5 every other day for 2 wk were tested for their ability to generate CTL in vitro. Two days after the last subcutaneous injection of thymosin, nude spleens were removed, mixed with normal BALB/c spleen cells, and placed into a mixed lymphocyte tumor culture (MLTC) against allogeneic RBL 5 tumor cells. After a 5-day incubation, cultures were tested for the presence of CTL in a 4-hr 51Cr-release assay. Spleen cells from thymosin-treated nude mice did not generate CTL but suppressed the ability of normal spleen cells to generate CTL in vitro. Characterization of the thymosin-induced nude mouse suppressor cells showed them to be Thy 1 positive, nonadherent, cyclophosphamide-sensitive T cells. These data demonstrate that some T cell maturation occurs in vivo under thymosin influence. However, the activity of these cells is initially limited to a regulatory function. These studies suggest that maturation of functional suppressor T cells occurs before CTL. Further immunologic manipulation appears to be necessary in order to induce CTL effector cells in nude mice.     

Persistent infection with mouse hepatitis virus of low virulence in nude mice.

A persisting type of infection with wasting syndrome was established in congenitally athymic nude mice after intraperitoneal inoculation with a mouse hepatitis virus which was not fully pathogenic for heterozygous haired littermates. From the liver, spleen, lymph nodes, and brain of most infected nude mice, the virus was detected at high titers during aperiod from 6 to 35 days postinfection, occurrence of degenerative and necrotic lesions being correlated with virus titers in these organs. The titer of serum neutralizing antibody remained undetectable or very low in most diseases nude mice, whereas some animals resisting the infection could produce antibody at a later stage. In heterozygous haired mice, some lesions were detectable at a very early stage of infection in the spleen and liver, but they seemed to disappear with a marked elevation of the neutralizing antibody titer. Nude mice were able to resist the virus infection when they had previously received transfer of thymocytes from weanling heterozygous littermates.     

IgG antibodies to asialoGM1 are more sensitive than IgM antibodies to kill in vivo natural killer cells and prematured cytotoxic T lymphocytes of mouse spleen

IgG and IgM antibodies, which were isolated from the anti-asialoGM1 (GA1) serum, had different effects against natural killer (NK) and prematured cytotoxic T cells (CTLs) in in vivo administration and in in vitro treatment. In in vitro treatments, the IgM antibody killed NK cells of nude mouse spleen in the presence of complement (C') 12 times more potently than the IgG antibody did, and either antibody with C' killed pre-CTL. In in vivo administrations, only the IgG antibody was effective in diminishing NK activity of the nude mouse spleen cells and in suppressing antigen-specific CTL induction from primed spleen cells by in vitro stimulation with X-irradiated tumor cells. The IgM antibody was not effective at all in either system. The in vivo effect of the IgG on NK activity was blocked by preadministration with silica or carrageenan but not by that with cobra venom factor (CVF). These results indicate that in vivo administration of anti-GA1 antiserum leads to macrophage-mediated depletion of CTL precursors as well as NK cells.     

Splenic suppressor cells and cell-mediated cytotoxicity in murine schistosomiasis.

An impairment of the capacity to generate alloantigen-specific cytotoxic T lymphocytes (CTL) was observed in mixed lymphocyte cultures (MLC) established with spleen cells from mice infected with Schistosoma mansoni. This impairment, which was observed as early as the eighth week of infection, could be abrogated by the fractionation of spleen cell suspensions by the carbonyl iron/magnet method prior to the establishment of MLC. Cocultivation of normal spleen cells with increasing numbers of splenocytes from S. mansoni-infected syngeneic mice resulted in a dosage-dependent suppression of CTL generation. This "infectious suppression" was not sensitive to antiserum against mouse thymic lymphocyte antigen (MTLA). The present studies suggest the role of a macrophage rather than a T cell as the suppressor cell in this model of cell-mediated immunity in schistosome-infected mice.     

Suppressor T cells in thymus-reconstituted nude mice: regulation of mitogen-induced transformation

A population of glass-wool-adherent splenic cells which could suppress the response of other spleen cell populations to T-cell mitogens was isolated from thymus-reconstituted nude mice. Such adherent cells were characterized as sensitive to anti-Thy 1.2 and complement treatment. Glass-wool-adherent cells from athymic mice do not have suppressor activity to self or normal littermate NAC; however, these mice possess precursor suppressor cells as demonstrated by isolation of glass-wool-adherent T regulatory cells in thymus-grafted nude mice. Such cells are generated in either freshly obtained or in vitro cultured thymus. Evidence for suppressor T cells of host genotype was supported by their sensitivity to host-specific anti-Thy serum treatment as well as their generation in alymphoid thymus grafts. Prior anti-Thy 1.2 treatment of GAC partially removed the suppressor activity: however, macrophages and B lymphocytes were shown not to be secondary regulatory cells or suppressor mediators, thus mature T lymphocytes with low amounts of Thy 1.2 antigen may be responsible for this residual suppression. Further characterization of GAC indicates that active cell growth is required for their regulatory function, as irradiation removed the suppressor activity. This report provides evidence for the presence of a T-lymphocyte subpopulation which has a regulatory function and requires a thymus in the generation of these cells.     

Mechanisms of the adjuvant effect of nystatin on in vitro antibody response of mouse spleen cells: indication of nystatin as a B-cell mitogen and as a stimulant for polyclonal antibody synthesis in B cells.

Adjuvanticity of nystatin, one of the polyenic antifungal antibiotics having as its primary target the membrane sterol of eukaryotic cells, was investigated by examining its effect on several functions of mouse spleen cells relevant to immunological phenomena in vitro. Nystatin was found to stimulate significantly DNA synthesis in thymus-independent (B) cells but not in thymus-dependent (T) cells. Like the other B-cell mitogens such as bacterial lipopolysaccharide (LPS), nystatin elicited nonspecifically polyclonal antibody synthesis in mouse spleen cell cultures, and also restored antibody response of T cell-deficient spleen cells of congenitally athymic nude mice to heterologous erythrocytes (RBC; thymus-dependent antigen). Thus, nystatin and LPS appeared to cause similar changes in the functions of spleen cells relevant to immunological events. However, antagonism but no additive effect in the adjuvanticity was revealed between the two adjuvants. As an interesting finding, the polyclonal generation of anti-RBC antibody-forming cells (AFC) in the spleen cell cultures by stimulation with B-cell mitogen, i.e., either nystatin or LPS, was not inhibited at all by inclusion of any anti-RBC antiserum, whereas, as is well known, the generation of AFC by stimulation with the antigen was specifically suppressed by the corresponding antiserum, indicating a difference in the genesis between the mitogen-induced AFC and the antigen-induced AFC.     

Activation of suppressor T cells by low-molecular-weight factors secreted by spleen cells from tumor-bearing mice

The spleens of mice bearing large M-1 fibrosarcomas have been shown to contain several populations of cells which nonspecifically suppress antibody synthesis by cocultured normal spleen cells. It has now been shown that the spleens of tumor-bearing mice also contain inducer cells which secrete soluble factors capable of activating suppressor T cells from unprimed precursor cells. The activated suppressor cells are Thy 1+, Lyt 1+2+ and secrete a soluble suppressive factor. They inhibit the in vitro generation of antibody-forming cells by cocultured normal spleen cells stimulated by T-cell-dependent antigens. They do not, however, suppress the antibody response to T-cell-independent antigens and do not inhibit antibody synthesis by cocultured nude mouse spleen cells cultured with T-cell-dependent antigens and exogenous helper factors. In addition, suppression is blocked if conditioned medium containing T-cell growth factors is added to the suppressor cell assays. These data suggest that cells in the spleens of tumor-bearing mice secrete inducing factors which activate suppressor cells. These activated suppressor cells in turn secrete soluble suppressor factors which inhibit antibody synthesis, possibly by interfering with the synthesis or release of T-cell growth factors.     

Direct evidence for sialic acid oxidation in periodate-induced lymphocyte proliferation.

Normal mouse spleen cells treated with periodate were stimulated to undergo blastogenesis. In contrast spleen cells from nude mice did not respond to periodate. Such a treatment of normal and nude spleen cells led to the oxidation of the cell surface sialic acid residues with formation of N-AN8. Prior treatment with neuraminidase of normal spleen cells greatly impaired their capacity to respond to periodate activation with a decrease in the amount of N-AN8 formed.     

Soluble suppressor activity of concanavalin A-activated spleen cells on B-lymphocyte colony formation in vitro.

Supernates from concanavalin A (Con A)-activated mouse spleen cell cultures suppress the formation of B-lymphocyte colonies (BLC) in soft agar culture by 30 to 95%. Con A-induced BLC suppressive culture supernates can be heated at 80 °C for 1 hr without losing activity. The BLC suppressive activity is eliminated totally by trypsin treatment and partly by treatment with β-galactosidase. Activity is unaffected by treatment with DNAse, RNAse, and α-glucosidase. By ultrafiltration the BLC suppressive factor(s) was shown to have a molecular weight greater than 300,000. These data suggest that BLC suppression is mediated by a protein-carbohydrate complex. BLC suppression was obtained when normal spleen cells were preincubated in Con A-activated supernates for only 1 hr at 37 °C. BLC suppressor activity was absent in the supernatant fluid of Con A exposed anti-θ-treated spleen cells, nonadherent spleen cells, extensively washed spleen cells, and spleen cells from nude (athymic) mice suggesting that cells responsible for Con A-induced BLC suppression are adherent, fragile cells of the T lineage. Con A-activated spleen cell supernates do not suppress colony formation in soft agar by normal mouse granulocyte-macrophage precursors, by plasmacytoma cells, T-lymphoma cells, or by carcinoma cells. However, colony formation by Abelson's murine leukemia virus transformed B-lymphoma cells was suppressed by 95% suggesting a relationship between this immature B-lymphoma line and B-lymphocyte colony-forming cells. Con A-activated spleen cell supernates do not suppress lymphocyte activation in liquid culture by phytohemagglutinin, Con A, or lipopolysaccharide. Heat-treated supernates—which inhibited BLC development by 90–95%—did not suppress the plaque formation by spleen cells immunized in vivo or in vitro by sheep red blood cells.     

Demonstration of antigen-specific suppressor cells in unresponsive nude mice: a model for the induction of antigen-specific suppressor cells

Nude spleen cells were rendered incapable of giving an in vitro response in the presence of TRF by pretreatment of nude mice with antigen, followed 1 week later by T-cell injection. It is shown that this unresponsiveness is caused by antigen-specific suppressor cells which affect not only the stimulation of nude spleen cells but also the activation of memory cells and the production of a T-cell-replacing factor. The appearance in nude mice of suppressing activity rather than helper activity, after administration of T cells, is dependent on the sequence of treatment. These results suggest a model for the induction of antigen-specific suppressor cells by activated B cells.     

Genetic control of effector cell characteristics: con A-induced suppressor cells carry H-2 I region encoded determinants.

Activation of splenic lymphocytes with Con A leads to the formation of suppressor cells capable of interfering with the activity of several polyclonal B-cell-activating substances. Thus, these suppressor cells, or their products, most probably act directly on B cells. Suppressor cells could be recovered from the effluent cell population of nylon wool columns, and they were absent from the spleens of athymic nude mice. Furthermore, they were absent from the thymus of normal as well as cortison-treated mice. Cortisone treatment did not abolish the formation of Con A-induced suppressor cells in the spleen. Treatment of activated suppressor cells with antisera specific for distinct products of the H-2 I region revealed that they carried I-J cell surface antigens. We conclude that the suppressor cells in our test system, which unlike other Con A-induced suppressor cell populations have a direct effect on B cells, had antigenic characteristics similar to those previously described for I-J carrying suppressor cells.     

The role of Ia antigens in the activation of T cells by concanavalin A: an evidence for the species restriction between T cells and accessory cells

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.     

Natural suppressor (NS) activity from murine neonatal spleen is responsive to IFN-gamma

Natural suppressor (NS) cell activity is the ability of apparently unprimed "null" cells to nonspecifically suppress immune responses. Previously we have shown that NS cell activity from the spleens of mice undergoing chronic graft-vs-host disease (GVHD) is enhanced in vitro by activated T cell signals (e.g., Con A supernatant [CAS]). Here we asked if the naturally occurring suppressor activity found in the neonatal mouse spleen is caused by NS cells, and if so whether this NS activity is also responsive to T cell signals. Finally, we wanted to identify the material in the CAS to which the NS cells respond. Spleen cells from (BALB/c X B10.D2)F1 neonates contain potent, genetically unrestricted suppressor activity toward normal mitogen responses. The cells responsible for this suppression are nonadherent, Thy-, Ig- and are thus by definition NS cells. Neonatal spleen NS cells suppress the indicator Con A response of all mouse strains tested, but their behavior with regard to LPS responses is different. They significantly inhibit the indicator LPS response of allogeneic strains, but are less inhibitory of LPS-stimulated syngeneic (BALB/c X B10.D2)F1 and parental strains. However, the addition of CAS to these latter cultures enhances the NS inhibition of the LPS response to the level of suppression seen with a Con A response. Two lymphokines were able to replace the CAS. Recombinant interferon-gamma (rIFN-gamma) closely mimics the activity found with whole CAS, with low concentrations (1 U/well) being capable of enhancing the neonatal NS activity to near-maximal levels. Recombinant interleukin 2 (rIL 2) is also capable of stimulating the neonatal NS activity to near maximum. However, the rIL 2 must be added at much higher concentrations, taking greater than 50 U/well to get maximum activation of NS suppression. The addition of anti-IFN-gamma antiserum to these LPS suppression assays removes the ability of CAS to activate the neonatal NS cells. Anti-IFN-gamma antiserum also removes the ability of rIL 2 as well as rIFN-gamma to activate the NS cells. It thus appears that the rIL 2 is working by its ability to stimulate IFN-gamma production. Anti-IFN-gamma also removes the ability of the neonatal NS cells to suppress a Con A response. Therefore, it appears that neonatal splenic NS cells respond to, and are activated by, IFN-gamma to carry out their suppressive activity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Tumorigenicity of persistently infected tumors in nude mice is a function of both virus and host cell type.

Although BHK-21 cells persistently infected with wild-type vesicular stomatitis virus (VSV) are sensitive to natural killer (NK) cells and do not form tumors in athymic nude mice, BHK-21 cells persistently infected with a previously isolated mutant virus (VSV-P) are resistant to NK cells and form tumors in nude mice. We used this VSV-P mutant to persistently infect HeLa cells and mouse tumor cell lines. A mouse mastocytoma line (P815) persistently infected with VSV-P was similar to BHK-21 cells in that it was resistant to NK cell lysis and formed tumors in nude mice. However, neither HeLa cells nor mouse myeloma lines persistently infected with VSV-P were resistant to NK cell lysis in vitro, and neither formed tumors in nude mice. Rejection by nude mice of HeLa cells and mouse myeloma cell lines persistently infected with VSV-P could be ablated by rabbit antiserum to asialo-GM1, implicating NK cells in the in vivo rejection of these persistently infected tumors. These results suggest that NK cell recognition and killing of virus-infected cells in vivo and in vitro depend upon genetic contributions from both the virus and the host cell.     

Modulation of natural cytotoxicity by alloantibodies: III. Augmentation of natural killer activity by alloantisera directed against K,D, and I regions of the murine H-2 complex

Natural killer activity of mouse spleen cells toward a human myeloid leukemia cell line, K562, can be enhanced by alloantisera directed against individual antigens in the H-2 region. By using a panel of 13 antisera (8 directed against antigens in the K and D regions and 5 directed against antigens in the I region) and four strains of mice (C57BL/6J, CBA, DBA/2, and A/J) it was found that certain antisera would stimulate target cell lysis by spleen cells only if the antisera had specificity for antigens which were a part of the haplotype represented on the spleen NK effector cells. Anti Ia antisera could stimulate the anti K562 NK activity of nude mouse spleen cells which lack mature T cells. Depletion of B cells and macrophages from nude spleen cells, by passing through a nylon-wool column also did not abolish the effect of anti-Ia antiserum. It appears likely therefore that the anti-Ia antibodies exert this effect directly on NK cells and that Ia antigens may be expressed on NK cells. Since the antisera directed against different antigens in H-2 complex irrespective of subregion specificity (K, D, or I) stimulated the NK activity of mouse spleen cells, the phenomenon offered an interesting method for testing the presence of a given alloantigen on mouse spleen cells. Log-dose response curves for the augmentation of lysis induced by appropriate alloantisera were linear over a dilution range of 1:320 to 1:5120. By using the dose-response curves, potency ratios of two preparations of antisera (directed against antigen 33 of the K region) could be successfully determined. Besides the K562 cell line, many human lymphoblastoid cell lines could also be used as target cells in this assay system.     

The immunoregulatory role of bone marrow. II. Characterization of a suppressor cell inhibiting the in vitro antibody response.

The addition of bone marrow cells (BMC) to spleen cell cultures suppressed the antibody response in a dose-dependent manner. This suppression required viable cells. Treatment of BMC with anti-thymocyte serum did not affect the suppressive activity and BMC, but not spleen cells, from nude mice inhibited the antibody response to the same degree as marrow from normal littermates. BMC which had been depleted of macrophages with antimacrophage serum or carbonyl iron showed increased suppressor activity. Furthermore, fractionation of BMC by velocity sedimentation and resetting revealed the suppressor cell to be a medium-to-large Fc receptor-positive lymphocyte. Absence of detectable B or T cell markers on the suppressor cell indicates this cell to be an Fc-positive null lymphocyte, possibly a precursor cell, which inhibits the response of mature lymphocytes     

Classification of interferons with antibody to immune interferon

Mouse immune Interferon, induced by the T-cell mitogen staphylococcal enterotoxin A (SEA), was partially purified and used to immunize rabbits. The resulting antiserum neutralized all immune interferon preparations tested, including interferon induced in vitro by SEA, concanavalin A, phytohemagglutinin P, and pokeweed mitogen, and in mixed lymphocyte cultures. Interferon produced in vivo with specific antigen was also neutralized. The antiserum was equally potent against all these interferon preparations. The serum did not neutralize any virus-type interferon preparation tested, but immune interferon induced by SEA in athymic nude mouse spleen cells was neutralized. The neutralizing activity was precipitable by 33% ammonium sulfate, and was not removed by absorption of the serum with mouse cells. The data suggest that immune interferons produced under diverse conditions are antigenically the same or closely related.     

Mechanism for the suppression of gamma-interferon responsiveness in mice after thermal injury

As reported previously, gamma-interferon production was decreased after the administration of inducers to thermally injured mice as compared with noninjured controls. Similarly, spleen cells from injured mice had decreased ability to produce interferon in vitro after stimulation with inducers. The present study demonstrated that the decrease in interferon production was associated with the presence of suppressor cells in the spleen of burned mice that were capable of inhibiting interferon production by normal splenic lymphocytes in vitro. Passive transfer of spleen cells containing suppressor cell activity derived from injured mice induced suppression in normal mice, and the time of the appearance of suppressor cell activity in injured mouse spleens closely approximated the time of the appearance of the suppression of interferon production observed in mice after thermal injury. The suppressor cells were characterized as a population of macrophages by the following: they adhered to plastic surface and could be removed from spleen cells by carbonyl-iron treatment; treatment of plastic-adherent cells with anti-Thy-1.2 and anti-mouse immunoglobulin antisera followed by complement failed to abrogate the suppression produced by these cells.     

Synergy between subpopulations of normal mouse spleen cells in the in vitro generation of cell-mediated cytotoxicity specific for "modified self" antigens.

Responding lymphoid cells cultured in vitro with irradiated trinotrophenyl (TNP)-modified syngeneic spleen cells develop direct cell-mediated cytotoxicity which is specific for target cells bearing both the TNP moiety and histocompatibility determinants of the modified sensitizing cell. Two subpopulations of normal mouse spleen cells have been shown to synergize in the in vitro generation of specific cell-mediated cytotoxicity to these "modified self" antigens. The synergizing populations are nylon wool column-adherent and column-nonadherent fractions of normal mouse spleen. When mixtures of these two cell populations are cultured in vitro with irradiated TNP-modified syngeneic spleen cells, greater cytotoxicity is generated in the two populations sensitized separately. The synergizing cell in the column-adherent population is resistant to lysis by rabbit anti-mouse brain serum, is distinct from the cytotoxic effector T lymphocyte, and is unresponsive to phytohemagglutinin; its synergizing function could not be replaced by peritoneal cells. These results suggest that it is a non-T cell which may be distinct from the macrophage.     

Inhibition of antibody-dependent cell-mediated cytotoxicity (ADCC) by antiserum raised against brain tissue

Treatment of mouse spleen cells with a rabbit anti-mouse brain (RAMB) antiserum markedly suppressed antibody-dependent cell-mediated cytotoxicity (ADCC) on trinitrophenyl-coupled sheep erythrocyte targets. This inhibitory activity of RAMB antiserum was complement independent, absorbable with mouse brain tissue, and appeared to be separable from the anti-Thy-1 activity of this serum. Absorption studies indicated that various T- and B-lymphocyte cell lines as well as macrophage-like cell lines are not able to absorb the inhibitory activity of RAMB antiserum. In contrast, thymocytes and spleen cells, as well as the neural cell line, PC12, a chromocytoma derived from rat adrenal medulla, were capable of absorbing the inhibitory activity to some extent, suggesting that antigens characteristic for ADCC effector cells can be found on these cell populations.