Nature of neonatal splenic suppressor cells in the mouse

Due to the controversy in the recent literature concerning the T-cell nature of the neonatal mouse splenic suppressor cell, we have reexamined the nature of these cells at different stages after birth. With the use of monoclonal anti-Thy antibody we can detect T-cell products on spleen suppressor cells from BDF₁ mice at 3, 4, 5, 6, and 7 days of age. The suppressor cells are assayed by their ability to inhibit mixed-lymphocyte reactivity, which is an in vitro example of cell-mediated immunity. The neonatal spleen suppressor cells also carry products of the Ly 1 and I-J locus. Neonatal thymectomy results in a premature decrease of suppressor cell activity. These data suggest that the mouse neonatal splenic suppressor cell is a short-lived Ly 1⁺, I-J⁺ T cell.     

Further studies on suppressor cell activity in the spleen of neonatal mice.

Spleen cells from newborn BDF₁ and C57BL/6 mice are tested for suppressor activity in mixed lymphocyte culture. The added spleen cells, treated with mitomycin, suppress both cell proliferation and the generation of cytotoxic cells. Thymus cells from newborn mice have little suppressor activity. Suppressor cells act across an allogeneic barrier, are cortisone-resistant, XR-resistant, T-cells and their activity is inversely correlated to the MLC reactivity in the spleen of the young animal. Incubation of neonatal suppressor cells on macrophage monolayers reduces suppressor cell activity and points to the possible importance of the macrophage in controlling immunological maturation.     

Suppressor T cells derived from early postnatal murine spleen inhibit cytotoxic T-cell responses

Postnatal T-suppressor cells have been detected in a number of experimental systems. They have been shown to inhibit humoral responses, proliferation in a mixed-lymphocyte reaction and the induction of killer cells. The suppressor function observed in the postnatal mouse does not appear to be antigen specific and its ontogeny may be influenced by other cell types and by serum factors such as α-fetoprotein. We have detected a nonadherent, radioresistant splenic T cell present in neonatal mice ranging in age from 1 to 9 days which can nonspecifically suppress killer cell induction. This suppressor cell must be cultured in vitro in order to function, but it does not require alloantigen to be induced. Adult spleen cells tested in the same system yield antigen-specific T-cell suppression. Our results suggest that the nonspecific suppressor detectable in 1- to 9-day-old mice disappears in adult life, and is replaced by antigen-specific suppressors. The biological role of these suppressors is discussed.     

Demonstration of active suppressor cells in spleens of young NZB mice

NZB mice, a strain prone to the development of autoimmune disease, have during the first 2 weeks of life suppressor cells in their spleens which can in coculture with adult spleen cells suppress the antibody response to sheep red blood cells (SRBC) generated in culture by the adult cells. The suppressive activity of spleen cells from NZB mice in the first week after birth is similar to that of spleen cells from 4-day-old C57BL/6 mice, a strain which does not spontaneously develop autoimmune disease. As in “normal” strains of mice, suppressor cell activity in NZB mice is diminished at 2 weeks and undetectable at 3 weeks of age. The data indicate that there is no defect inherent in the suppressor cells detected in the spleens of newborn and young NZB mice and suggest that the development of autoimmune responses does not result from a lack of suppressor cells in the young animals.     

Suppressor factor produced by neonatal mouse spleen cells

Spleen cells from 5- to 6-day- old BDF₁ mice produce a soluble suppressor factor (SF) when cultured for 2–4 days in the presence of 10% fetal calf serum. This suppressor factor inhibits the mixed lymphocyte reactivity of adult mouse spleen cells as well as the in vitro generation of cytotoxic cells. The SF which is not H₂-restricted or antigen specific is most effective when added in the early phase of the culture period. The SF is resistant to heat and uv treatment and appears to consist of a large and small component. It is resistant to treatment with pronase or trypsin. The SF appears to be produced by neonatal spleen cells which are not adherent to plastic or Sephadex G-10 and are insensitive to treatment with anti-Thy 1.2 and complement. Incubation of SF with peritoneal exudate cells reduces suppressor activity.     

Effect of injection of adult mouse peritoneal macrophages on suppressor cell activity in neonatal mice

Injection of adult mouse peritoneal exudate cells into newborn mice results in a premature decrease of splenic suppressor cell activity in the neonates. The effect becomes apparent 4–5 days after ip injection of 10–15 × 10⁶ thioglycollate-induced peritoneal exudate cells into mice on the day of birth. The macrophage in the peritoneal exudate is the responsible cell type. The effect is not H-2 restricted or strain limited. Heat-killed peritoneal exudate cells or peritoneal cells from unstimulated donors can also decrease neonatal suppressor cell activity prematurely. Adult spleen cells, injected into neonatal mice, do not affect suppressor cell activity. The data are discussed in light of the hypothesis that macrophages control suppressor activity in neonatal mice and that an increase in the number and/or function of macrophages shortly after birth results in a decrease in the number and/or function of suppressor cells, allowing for immunological competence to emerge.     

Induction of T15-specific suppressor T cells in mice with the CBA/N defect by neonatal injection with anti-T15 idiotype antibody

Mice with the CBA/N defect (xid) are unresponsive to phosphorylcholine (PC), To determine whether idiotype-specific suppressor T cells can also be generated in these defective mice, defective (CBA/N X BALB/c)F1 male and nondefective (CBA/N X BALB/c)F1 female or (BALB/c X CBA/N)F1 male mice were neonatally injected with antibodies specific for the major idiotype of anti-PC antibody, i.e., anti-TEPC-15 idiotype (T15id) antibody. Suppressor cell activity was examined by co-culturing spleen cells from neonatally treated F1 mice with spleen cells of normal nondefective F1 mice in the presence of antigen. Spleen cells from defective (CBA/NM X BALB/c)F1 mice treated with anti-T15id antibody demonstrated a level of suppressor activity (greater than 83% suppression) comparable to that of similarly treated nondefective F1 mice. This suppression was specific for the T15id of anti-PC response, and a Lyt-1-2+-bearing T cell population appeared to be responsible for the active suppression. These suppressor T cells recognized T15 but not PC, based on a functional absorption test. These results indicate that the CBA/N defects, including the deficiency in the anti-PC response by B lymphocytes and a possible T cell defect, do not influence the generation of T15id-specific suppressor T cells by neonatal injection with anti-T15id antibody.     

Stimulation of suppressor cells in the bone marrow and spleens of high dose cyclophosphamide-treated C57Bl/6 mice

Systemic administration of a single dose (300 mg/kg) of cyclophosphamide (Cy) induced the appearance of a population of suppressor cells in the bone marrow and spleens of mice. Suppressor cells were assayed by their capacity to inhibit the concanavalin A (Con A) blastogenesis or the mixed-lymphocyte response of normal C57Bl/6 spleen cells. Cy-induced bone marrow (Cy-BM) suppressor cells were present as early as 4 days following Cy therapy and their activity gradually decreased over the next 2 weeks. Cy-induced splenic (Cy-Sp) suppressor cells were maximally present on Days 6 through 10 following Cy therapy. Studies were performed to characterize the suppressor cells of bone marrow obtained 4 days after Cy treatment and of normal bone marrow (N-BM). Some suppressor activity was present in normal bone marrow. N-BM suppressor cells resembled cells of the monocyte/macrophage lineage in that they were slightly adherent to Sephadex G-10, sensitive to L-leucine methyl ester (LME), and insensitive to treatment either with anti-T-cell antibody and complement or with anti-immunoglobulin antibody and complement. Their suppressive activity was abrogated by incubation with either indomethacin or catalase. Cy-BM suppressor cells were also resistant to treatment with anti-T-cell and anti-immunoglobulin antibody and complement but were not adherent to Sephadex G-10 and not sensitive to LME. Their suppressive activity was partially eliminated by indomethacin alone or in combination with catalase. We conclude that Cy chemotherapy induces the appearance of a population of immune suppressive cells and that these cells appear first in the bone marrow and subsequently in the spleen.     

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     

Suppressor factor from tumor-allosensitized spleen cells--its effect on in vitro proliferation of tumor cells and in vivo skin allograft survival

C57BL/6 mice are sensitized ip with allogeneic P-815 mastocytoma cells. Fifteen days later the spleen cells of the sensitized mice are used in the production of suppressor factor or treated with mitomycin and used as suppressor cells. Sensitized spleen cells incubated with the specific alloantigen (DBA/2 m-treated spleen cells) release suppressor factor (SF)² which inhibits cell proliferation in mixed lymphocyte culture (MLC) as well as the in vitro generation of cytotoxic cells (CML). SF is most effective when added eary during MLC. SF also inhibits mitogen responsiveness of normal spleen cells. In addition to inhibiting lymphocyte function in vitro, suppressor cells as well as SF inhibit the in vitro proliferation of tumor cells. This inhibition is specific for the tumor to which the suppressor cells are induced. The inhibition of tumor cell proliferation is not due to the presence of cytotoxic cells in the spleen of the tumor-allosensitized mice. Suppressor cells from neonatal mice do not inhibit the in vitro proliferation of tumor cells. SF injected iv into C57BL/6 mice decreases the mixed lymphocyte reactivity of the host spleen cells and decreases the ability of the host to reject skin allografts. We interpret these data to suggest that tumor-allosensitized spleen cells, and the SF they produce, not only affect lymphocyte function but also inhibit tumor cell proliferation. This dual effect of suppressor cells could be an important part of the immune surveillance against tumors.     

Spontaneous IFN-beta production. A common feature of natural suppressor systems

Natural suppression has been described in several immunologic systems in which splenocytes are not only incapable of generating an immune response, but are also able to non-specifically suppress normal splenocyte reactivity. Natural suppressor cells exist in such transitory immune deficiency states as graft vs host disease, irradiation recovery, and during neonatal development. We have found that when splenocytes from each of these three systems were placed in culture, IFN was produced spontaneously without stimulus. This spontaneous IFN production was augmented by the addition of IL-2 or granulocyte-macrophage-CSF to cell cultures. However, these lymphokines induced little or no IFN from normal splenocytes. This unusual IFN production is especially interesting since all samples have been typed to be IFN-beta. Additionally, greater IFN-beta was produced by these spleen cells during active suppression of the MLR of normal spleen cells. In fact, antibody to IFN-beta was shown to partially reverse the natural suppression mediated by graft vs host disease splenocytes in the MLR. Thus, IFN-beta production correlates extremely well with natural suppressor states. As mice begin to recover normal immune responses in all three systems, IFN-beta ceases to be produced spontaneously in culture. These findings establish a previously undescribed IFN-beta-producing splenic phenotype and suggest that IFN-beta may contribute to the immunoregulation of natural suppressor systems.     

Influence of 2'-deoxyguanosine upon the development of DTH effector T cells and suppressor T cells in vivo

Subcutaneous (s.c.) immunization of mice with allogeneic spleen cells can induce delayed-type hypersensitivity (DTH) to both major and minor histocompatibility antigens. Intravenous immunization with allogeneic spleen cells, however, induces a poor state of DTH. Furthermore, i.v. immunization with allogeneic spleen cells, especially if they have been irradiated, induces suppressor T lymphocytes. These suppressor T cells are capable of suppressing the host-vs-graft (HvG) DTH reactivity that normally arises after s.c. immunization. Moreover, they can suppress the development of anti-host DTH effector T cells during graft-vs-host (GvH) reactions. These models for HvG and GvH DTH reactivity were used to study the influence of 2'-deoxyguanosine (dGuo) and guanosine (Guo) on the generation of DTH-reactive T cells and suppressor T cells in vivo. It was found that daily i.p. administration of 0.01 mg dGuo to mice immunized i.v. partially prevented the generation of suppressor T cell activity, whereas daily administration of 0.1 or 1 mg dGuo resulted in a complete abolition. Administration of dGuo has no effect on the anti-host DTH reactivity by spleen cells from nonsuppressed donors except for when a daily dose of 10 mg is administered. This dose proved to be toxic for precursors of DTH effector T cells. Daily i.p. injection of Guo had no effect on the generation of suppressor T cells nor on the generation of DTH effector T cells. The effect of dGuo was found to be due to a direct effect on suppressor T cells and not to the induction of contrasuppressor cells. These data suggest a differential sensitivity of DTH-reactive T cells and suppressor T cells for dGuo. Because suppressor T cells and DTH-reactive T cells require proliferation for expressing maximal functional activity in the systems used, both cell types probably have different enzyme activities involved in the purine metabolism and similar deoxycytidine kinase activities, but have different nucleotidase (5'NT) activities, those in suppressor T cells being the lowest. If so, suppressor T cells will accumulate deoxyguanosine triphosphate, which causes an inhibition of the ribonucleotide reductase activity and thus of the DNA synthesis by these cells.     

T lymphocyte tolerance and early appearance of virus-induced cell surface antigens in Moloney-murine leukemia virus neonatally injected mice

Regression of Moloney-murine sarcoma virus- (M-MSV) induced sarcomas in normal adult mice is accompanied by generation of virus-specific cytotoxic T lymphocytes (CTL). However, when neonatal mice that were injected with Moloney-murine leukemia virus (M-MuLV carrier) were subsequently challenged as adults with M-MSV, the sarcomas did not regress nor did they generate CTL. This failure to produce CTL cannot be ascribed to nonspecific immunodepressive effects or to suppressor cell generation since M-MuLV carrier mice exhibit normal reactivity after allogeneic cell stimulation. Moreover, addition of M-MuLV-infected cells as the third party to cultures does not reduce activity of CTL from M-MSV immune mice. Since M-MSV and M-MuLV possess common antigens, the observed unresponsiveness was considered in relationship to induction of a T lymphocyte tolerance, which may follow introduction of foreign antigens at an early stage of development. In fact, it was observed that as early as 10 days after injection, thymus, lymph node, and spleen from M-MuLV carrier mice express virus-induced cell-surface antigens that not only are targets for M-MSV-immune CTL, but also induce in vitro a strong specific cytotoxic response. In addition, a cold target inhibition assay disclosed that the same antigens are shared by both M-MuLV infected and leukemia cells, even though they are less expressed on the surface of the former. The finding that the cytotoxicity of alloreactive lymphocytes from M-MuLV carrier mice is reduced after preincubation with M-MSV immune CTL confirms that virus infection does not bring about functional inactivation of lymphocytes. Finally, it was observed that virus antigen presence on lymphocytes from M-MuLV neonatally injected mice is closely related to subsequent leukemia development.     

Contact sensitivity to picryl chloride: the occurrence of B suppressor cells in the lymph nodes and spleen of immunized mice.

Mice were immunized for contact sensitivity and antibody production by painting the skin with picryl chloride. Lymph node and spleen cells taken 4 days later transferred contact sensitivity. However, cells taken at 7–8 days failed to transfer but were able to block the transfer by 4 day immune cells. These suppressor cells occurred in the regional lymph nodes, spleen and thymus. The suppressor activity of lymph node and spleen cells was due to B cells as shown by the effect of anti-θ serum and complement, nylon wool filtration and separation of EAC positive and negative cells by centrifugation on a discontinuous gradient. The transfer of fractions rich or poor in macrophages showed that the suppressor cell in the transferred population was not a macrophage. Separation using EAC rosettes suggested that B cells were responsible for the suppressor activity in the thymus.T cells isolated from the lymph nodes and spleen 7–8 days after immunization transferred contact sensitivity although the initial population was inactive. This indicates that passive transfer cells are present in the regional lymph nodes and spleen at later times after immunization but cannot be demonstrated because of the presence of suppressor B cells. However, no passive transfer cells were found in the thymus. The production of B suppressor cells required little or no T cell help and following immunization the spleens of reconstituted (B) mice were at least as active as control cells in causing suppression. There are several different suppressor cells which act in the picryl system and the B suppressor cells in immunized mice described here are distinct from the T suppressor cells in mice injected with picryl sulphonic acid.     

Suppression of IgE antibody production in SJL mice. V. Effect of irradiation and adult thymectomy on the suppression of IgE antibody production in SJL mice

Anti-DNP IgE antibody production was low and transient in SJL mice which were immunized with 1 microgram DNP-Nb and 1 mg A1(OH)3. The immunized SJL mice were irradiated (60-540 R) 1 day after challenge. A dose higher than 180 R induced enhancement of anti-DNP IgE antibody production as compared to nonirradiated control mice, suggesting the existence of irradiation-sensitive suppressor cells. Anti-DNP IgE antibody production was suppressed when immunized and irradiated SJL mice were injected with spleen cells from adult-thymectomized SJL mice. The donors of the spleen cells were thymectomized 2 or 4 months previously, and this suggests that the suppressor cells from unprimed mice are long-living T cells.     

BCG-induced suppressor cells. I. Demonstration of a macrophage-like suppressor cell that inhibits cytotoxic T cell generation in vitro.

Spleen cells obtained from mice 5 to 40 days after infection with viable BCG organisms (BCG-spleens) were found to be unresponsive in vitro to both mitogenic and alloantigenic stimuli. Moreover, suppressor cells could be demonstrated in the spleens from these infected animals. When spleen cells from BCG-infected mice were added to either syngeneic or allogeneic normal spleen cells, the mixtures neither proliferated nor developed cytotoxic activity when cultured with alloantigen or with concanavalin A (Con A). The development of unresponsiveness post-infection paralleled the onset of suppressive activity. Spleen cells obtained from mice given heat-killed BCG were neither suppressive nor unresponsive. The suppressive activity of BCG-spleen cells was associated with an adherent, phagocytic cell that lacked membrane-associated Thy-1 antigen. Removal of this cell by passage through nylon wool columns resulted in a cell population that was no longer capable of suppression and that responded normally to alloantigen and to Con A. It would thus appear that BCG infection results in the development of a "suppressor" macrophage-like cell population within the spleen. The role of this cell type in regulation of the immune response in BCG-infected animals is as yet undefined.     

Immunologic tolerance to HGG in mice. I. Suppression of the HGG response in normal mice with spleen cells or a spleen cell lysate from tolerant mice.

Adoptive transfer of spleen cells or spleen cell lysates from mice tolerant to human-gamma-globulin (HGG) specifically suppressed the response of normal syngeneic recipients to HGG. The suppressive activity could be transferred for over 100 days after tolerance induction. The suppression induced by both spleen cells and spleen cell lysate was found to be specific as evidenced by a normal response to a challenge with turkey-gamma-globulin or goat erythrocytes. The activity of the suppressive lysate could be removed by passing the material through an HGG immunoadsorbent column but not by passing it through an anti-HGG column or a BSA column. These results indicated that the factor had antigen specificity and was probably not antigen-antibody complexes. That this suppression was not due to a shifting of the kinetics of the antibody response has also been demonstrated. The antigen-specific suppressor factor in the tolerant spleen cell lysates was a protein with a m.w. of approximately 45,000 daltons. The kinetics of the appearance of both suppressor cells and suppressor factor were consistent with a mechanism of active suppression functioning in the maintenance of tolerance to HGG.     

Suppressor cells in mice infected with Trypanosoma brucei.

Within 2 to 3 days of infection with Trypanosoma brucei strain S42, the ability of spleen cells from infected CBA mice to mount a primary in vitro antibody response to sheep red blood cells (SRBC) is profoundly reduced, and suppressor cells are generated as detected by cell mixture experiments. Suppressor cell activity lies in the T and adherent cell compartments of spleens from infected mice, but not in the B cell compartment, although antibody responses to a thymus-independent antigen, DNP-Ficoll, are significantly reduced. Suppression of antibody responses of normal spleen cells depends on viable cells from infected mice. The trypanosome, itself, plays no direct role in suppression, and we have ruled out the possibility of antigenic competition as a mechanism of suppression. Our data is consistent with the model of suppressor T cells induced by concanavalin A mitogenesis. We hypothesize that trypanosome antigens may directly stimulate T cells with the concomitant release of factors with affinity for macrophage surfaces thus becoming suppressive for T and B cell responses.     

Augmentation of transfer of experimental autoimmune thyroiditis (EAT) in mice by irradiation of recipients

Experimental autoimmune thyroiditis (EAT) can be adoptively transferred to normal syngeneic recipients using spleen cells from susceptible strains of mice primed in vivo with mouse thyroglobulin (MTg) and lipopolysaccharide (LPS) following in vitro activation of spleen cells by culture with MTg. Irradiation of recipient animals markedly augments the severity of thyroiditis induced in this system. Irradiation of recipients does not alter the time course of the development of thyroiditis, nor does it alter the requirement for both in vivo priming and in vitro activation of spleen cells for the development of EAT. Spleen cells from EAT-resistant strains of mice (e.g., Balb/c) do not induce EAT in irradiated recipients. Irradiated recipients develop significant levels of anti-MTg antibodies while unirradiated recipients have little detectable antibody response. The augmenting effect of irradiation can be substantially reversed by transferring naive spleen cells to recipients prior to the transfer of MTg/LPS-primed in vitro-activated spleen cells. In addition athymic CBA/Tufts nude mice develop more severe EAT than CBA/Tufts nude/+ littermates following transfer of activated CBA/J spleen cells. These data suggest that natural suppressor cells may regulate the development of EAT at the effector cell level.     

Lymphocyte function in experimental African trypanosomiasis. III. Loss of lymph node cell responsiveness

The relationship between immunosuppression and suppressor cell activity in the lymphoid organs of animals with experimental African trypanosomiasis has been examined further. In the present study we measure the primary in vitro PFC response to SRBC by spleen and lymph node cells from Trypanosoma rhodesiense infected or drug-cured C57BL/6 mice. Passive transfer experiments with this culture system tested for the presence or absence of suppressor cells. We demonstrate that infected mice exhibit immunosuppression in the spleen cell population several weeks before becoming suppressed at the level of the lymph node cell populations. Although suppressor cells are present in immunosuppressed spleen cell populations, suppression of lymph node cell responsiveness was not attributable to suppressor cells detectable withi, lymph nodes. After Berenil treatment of terminally infected mice immunocompetence was restored gradually, first to the lymph node cells and subsequently to the spleen cell population. Recovery of spleen cell responsiveness was attributable to the loss of detectable suppressor cell activity within spleens. These results demonstrate that there is anatomical restriction of the suppressor cell population to trypanosome-infected mouse spleen and that loss of immunocompetence in the lymph nodes may be due to factors unrelated to suppressor cell effects.