Natural Suppressor Cells Research Articles

INDUCTION OF NATURAL SUPPRESSOR-LIKE CELLS FROM HUMAN ADULT PERIPHERAL BLOOD LYMPHOCYTES BY A K562-DERIVED FACTOR

K562-AC1, a subclone of the human myeloid leukemia cell line K562, secreted an inhibitor of PHA-stimulated human T cell growth into the culture supernatant (K562sup). Kinetics and absorption assays in vitro revealed that the factor acts on PBMC during the very early growth phase. PBMC gained potent suppressor activity against autologous T cell growth after preculture with K562sup for 3 days in a dose-related manner. HLA class II negative and nylon wool-nonadherent subsets possessed higher suppressor potential than unfractionated PBMC. Treatment of K562sup-precultured PBMC with mAb followed by complement-lysing showed that the responsible suppressors belong to null cells expressing CD3-, CD4-, CD19-, CD14-, CD11b-, but partially CD2+. Exogenous IL-2 exhibited a synergistic effect on the activity of the suppressors committed by K562sup. However, K562sup inhibited IL-2-activated lymphocytes from generating anti-K562 cytolytic activity. Macrophages inhibited the induction of the suppressors, which was restored by adding indomethacin and/or IL-2. In conclusion, K562sup has the ability to induce efficient suppressor cells from human adult PBL, which belong to null cells without NK/lymphocyte-activated killer activity and are similar to natural suppressor cells.

Heterogeneity of splenic natural suppressor cells induced in mice by treatment with cyclophosphamide.

Administration of high dose cyclophosphamide (CY, 200 mg/kg body weight) to adult mice induces transient, nonspecific suppressor activity in the spleen of treated animals. Characterization of the CY-induced natural suppressor (NS) cells which inhibit mixed lymphocyte reactions revealed a heterogeneous population of lymphocytes expressing the CD8 T cell marker and the B220 B cell marker, as well as cells bearing the granulocyte-monocyte marker CD11b. On a cell per cell basis the most potent of these suppressors were found to be positive for CD11b. Inhibitory activity was also detected in the CD8-, CD11b-, B220- compartment of CY-spleen, suggesting the presence of null NS cells. The fact that several phenotypically distinct cell populations contribute to the overall inhibitory effect of CY-spleen cells indicates that natural suppression defines an activity rather than a specific cell type. Interestingly, NS activity was observed to reside solely within the fraction of CY-spleen that is agglutinable with soybean agglutinin or wheat germ agglutinin, suggesting that expression of receptors for these plant lectins is a universal characteristic of CY-induced NS cells, regardless of their lineage. CY-spleen cell-mediated suppression of lymphoproliferative responses was found to be partially dependent on DNA synthesis and totally dependent on protein synthesis, but did not require cell-cell contact, indicating the production of soluble suppressor factor(s).

Prevention of diabetes and induction of non-specific suppressor cell activity in the BB rat by an immunomodulatory azaspirane, SK&F 106610.

Immunomodulatory azaspirane compounds have immunosuppressive activity in animal models of autoimmune disease such as adjuvant-induced arthritis and experimental autoimmune encephalomyelitis. The mechanism of action of azaspiranes appears to be the induction of antigen non-specific (natural) suppressor cell activity. In this study, we tested the azaspirane, SK&F 106610 in an animal model of autoimmune (type 1) diabetes, the BB rat. Oral administration of SK&F 106610 (15 mg/kg/day) to diabetes-prone BB rats, from age 30 days, significantly decreased diabetes incidence at 100 days from 80% (24 of 30 control rats) to 32% (10 of 31 drug-treated rats, P < 0.001). Protection from diabetes by SK&F 106610 was accompanied by decreased lymphocytic infiltration of the pancreatic islets (insulitis). No changes occurred in splenic T cell, B cell or macrophage subsets, or in proliferative responses to the mitogens lipopolysaccharide and concanavalin A (Con-A). Cell mixing experiments in vitro, however, revealed increased antigen non-specific suppressor activity (suppression of splenic lymphoproliferative response to Con-A) in spleens of SK&F 106610-treated rats. The suppressor cell activity was enriched in a low density fraction of splenic cells relatively depleted of T cells, B cells, macrophages and natural killer cells. These results indicate that the azaspirane compound, SK&F 106610 can prevent insulitis and autoimmune diabetes in BB rats and that these actions may be related to the activation of non-specific (natural) suppressor cells.

Transforming growth factor-beta is the major mediator of natural suppressor cells derived from normal bone marrow.

We previously reported that murine bone marrow cells activated by interleukin-3 (IL-3) or granulocyte-macrophage colony-stimulating factor (GM-CSF) had potent nonspecific natural suppressor (NS) cell activity. In the present study, we demonstrated that these activated NS cells released a soluble factor (or factors) capable of nonspecifically inhibiting T cell mitogenic responses. Consistent with the properties of transforming growth factor-beta (TGF-beta), treatment of the NS supernates with heat failed to denature the factor, and in fact significantly increased its suppressive activity. The NS suppressor factor strongly inhibited proliferation of the TGF-beta-sensitive tumor cell line, A549. Cytokine activation of suppressive activity correlated with the production of a 10- to 13-kDa protein, consistent with the size of TGF-beta and rIL-3 induced a sevenfold increase in TGF-beta transcription. Finally, neutralizing anti-TGF-beta antibody inhibited the suppressive activity of the supernates, indicating that TGF-beta was responsible for most, if not all, of the suppression expressed by these bone marrow NS cells.

Identification of natural suppressor cells in long-term renal allograft recipients.

We have previously reported an abnormal expansion of CD3+Leu7+ (CD57+) large granular lymphocytes in long-term renal allograft recipients. These cells lacked NK activity, T-helper activity and did not respond to T cell mitogens. The following studies were done in order to further define the functional characteristics of these cells. We sorted CD3+Leu7+ T cells from the peripheral blood of 45 recipients (all with good renal allograft function), and found that these cells suppress mixed lymphocyte culture responses and pokeweed mitogen-induced IgG secretion in a non-genetically restricted manner. PWM-induced IgG secretion assays are suppressed by 60-100%, and MLC responses are suppressed by 20-85% at a ratio of 1:10 CD3+Leu7+ cells to responder/effector cells. Supernatants from CD3+Leu7+ cell cultures are also suppressive. On the other hand, unsorted cells and non-CD3+Leu7+ sorted cells either enhance responses or produce less than 10% suppression under the same conditions. Patients who were tested more than once showed a relatively stable percentage and suppressive effect of their CD3+Leu7+ cells over an interval of 6-12 months. These nonspecifically suppressive CD3+Leu7+ large granular lymphocytes are similar in many ways to the natural suppressor cells that have been identified in hematopoietic tissues, in graft-vs.-host disease, and in the lymphoid organs of neonates.

Cyclophosphamide-induced suppressor cells in nude mice.

Lymphocytes regenerated after treatment with a high dose of cyclophosphamide (CY) were characterized in nude mice. Ten days after a single injection of 200 mg/kg CY into nude mice, regenerated spleen cells suppressed in vitro primary and second antibody production against sheep red blood cells. The CY-treated spleen cells exhibited normal natural killer (NK) activity, very low B and T cell content, but increases in cell surface charge [electrophoretic mobility (EPM)] and histamine receptors. The suppressor cells could not be removed by treatment with anti-Thy-1 plus complement (C), or treatment with antiasialo GM1 (aGM1) plus C, which abrogated NK activity. It was concluded that CY-treated spleen cells, which exhibited high EPM and histamine receptors, comprise the natural suppressor cells which are Ig-, Thy-1- and aGM-1.

Soybean agglutinin-positive natural suppressor cells in mouse bone marrow inhibit interleukin 2 production and utilization in mixed lymphocyte reactions.

Although natural suppressor (NS) cells resident in bone marrow (BM) have been the subject of intensive study, the exact nature and mode of action of these potentially important immunoregulatory cells are still uncertain. Here we show that NS cells with potent inhibitory effects on mixed lymphocyte reactions (MLRs) can be isolated from BM of normal adult mice by agglutination with the plant lectin soybean agglutinin (SBA). Complement-dependent lysis of SBA receptor-bearing BM cells with antibodies to asialoGM1, Mac-1, Thy-1.2, J11d.2, and 2C1 phenotypic markers reveals the presence of at least two distinct populations of BM NS cells. Most of the SBA-binding BM cells with NS capacity have the null phenotype and resemble hematopoietic stem cells, and some inhibitory SBA+ BM cells express the 2C1 marker found on pregnancy-associated splenic NS cells and the J11d.2 antigen characteristic of B cells and immature T cells. Results of positive selective experiments confirmed these findings. The mechanism of natural suppression was also studied. Evidence is presented that SBA+ BM cells exert NS activity in MLRs by interfering with the production and utilization of interleukin 2. Indomethacin does not relieve natural suppression associated with SBA+ BM cells, indicating that prostaglandin synthesis is not a requirement for inhibitory function. However, neutralizing antibodies to transforming growth factor beta (TGF-beta) partially reverse the suppression mediated by SBA+ BM cells, suggesting that some BM NS cells may act through the release of an immunosuppressive molecule related to TGF beta.

Cytokine regulation of bone marrow natural suppressor cell activity in the suppression of lymphocyte function

Natural suppressor (NS) cells, which nonspecifically suppress immune responses, are present in the spleen following exposure to radiation, chronic graft-versus-host disease, or cancer and in normal bone marrow. A model system is described which allows the study of cytokines activating and inhibiting NS cells, cytokines mediating NS activity, and NS effects on cytokine synthesis. Recombinant interleukin-3 (rIL-3) and granulocyte-macrophage colony-stimulating factor (rGM-CSF) efficiently activated NS cells present in normal bone marrow and were effective at concentrations as low as 5 U/ml. At high concentrations, GM-CSF, but not IL-3, did not activate NS cells. Recombinant interferon-γ (rIFN-γ) blocked the activation of bone marrow NS cells by rIL-3, but did not down-regulate NS cells once activated. The NS cells secreted one or more soluble suppressor factors, which blocked IL-2 synthesis and also inhibited IL-2-dependent T cell proliferation in the presence of excess IL-2.

LPS Activation of bone marrow natural suppressor cells

Lipopolysaccharide (LPS) from Salmonella typhosa was injected into C57B1/6 mice and the effect on bone marrow (BM) natural suppressor (NS) cell activity was examined. It was shown that injection of LPS, as low as 0.01 μg/g body weight, could enhance BM NS activity. The enhanced activity was apparent 24 hr postinjection, and returned to normal by Day 5. It was necessary to show that the enhanced suppression displayed characteristics of NS cells. The suppressor cell is Thy negative and can be found in low density Percoll fractions. Suppression was dependent upon interferon-γ and could be augmented by lymphokines that were contained in the supernatant of TH 2 helper cell. The data suggest that BM NS activity may be influenced in vivo during gram-negative sepsis.

Reactivity of monoclonal antibody 1E5.B5 with a novel phenotypic marker expressed on a murine natural suppressor cell subset.

Natural suppressor (NS) cells are antigen-nonspecific, MHC-independent immunoregulatory cells that are typically found in murine bone marrow (BM), newborn (NB) mouse spleen, and in splenic tissue of adult mice during pregnancy and following cyclophosphamide (CY) treatment. There has been a pressing need for the development of NS cell-specific monoclonal antibodies (mAb) since NS cells are generally described as null cells which lack the usual phenotypic markers of mature T cells, B cells, and macrophages. Here we present evidence that mAb 1E5.B5, which was raised in rats against murine splenic pregnancy-associated NS (SPANS) cells, recognizes a unique antigenic marker expressed by some, but not all, murine NS cells. In the presence of complement, mAb 1E5.B5 effectively eliminates SPANS activity, and diminishes NS activity of CY-treated spleen cells in mixed lymphocyte reactions (MLR). However, cytotoxic pretreatment with mAb 1E5.B5 had minimal effects on NS activity of BM and NB spleen cells. We also show that pregnancy spleen cells and CY-spleen cells with moderate NS activity in MLR can be positively selected for by "panning" with mAb 1E5.B5. In contrast, only weakly inhibitory cells are isolated from BM and NB spleen by this procedure. Cellular ELISA and flow cytometry confirm that mAb 1E5.B5 has specificity for pregnancy spleen cells and CY-spleen cells, as well as for NB spleen and BM cell preparations. Western blot analysis reveals that mAb 1E5.B5 reacts with a novel 50 kDa NS cell-associated antigen which we have termed NS-1. The NS-1 antigen is not present on other null cells such as natural killer (NK) cells and natural cytotoxic (NC) cells since cytotoxic pretreatment of pregnancy spleen cells with mAb 1E5.B5 does not affect antibody-dependent cell-mediated cytotoxicity, NK or NC activity.

Pregnancy-associated suppressor cells in mice: Functional characteristics of CD3 +4 −8 −45R + T cells with natural suppressor activity

Natural suppressor (NS) cells are MHC-unrestricted regulatory cells with non-specific inhibitory activity for immune responses. In adult mice, NS cells are characteristically found in bone marrow and in splenic tissue following total lymphoid irradiation and cyclophosphamide treatments. Recently, we have shown that the spleens of pregnant mice harbour a population of lymphocytes which resemble NS cells in terms of phenotype and inhibitory activity. In this study, we use positive and negative selection techniques to further characterize splenic pregnancy-associated NS (SPANS) cells as predominantly ‘double negative’ T cells (CD3 +4 −8 −) bearing receptors for the lectins wheat germ agglutinin and soybean agglutinin, as well as expressing CD45R and the heat-stable J11d.2 antigen. Taken together, these findings lead us to conclude that SPANS cells belong to an immature T cell lineage. In keeping with their T cell phenotype, SPANS cells do not express the natural killer (NK) cell-specific markers NK2.1 and asialoGM1 and do not mediate lytic activity against NK-sensitive YAC-1 cells, although natural cytotoxic activity against WEHI-164 cells was found to co-purify with SPANS cells. Suppressive activity of SPANS cells in mixed lymphocyte reactions (MLR) is abolished by treatment with mitomycin C, suggesting that natural suppression in this system is a proliferation-dependent phenomenon. Preincubation of SPANS cells with conditioned medium from Con A-stimulated T cell cultures results in augmented NS activity, indicating that SPANS cells respond to T cell signals. Our data suggest that SPANS cells mediate suppression via the elaboration of a soluble suppressor factor since SPANS cells do not require cell-cell contact to mediate suppression and supernatants from short-term cultures of SPANS cell-enriched SBA + pregnancy spleen cells inhibit MLR. We believe that SPANS cells may be important in regulating hematopoiesis and maternal anti-fetal immunity during murine pregnancy.

Suppressor function of liver mononuclear cells isolated during murine chronic graft-vs-host disease: II. Role of prostaglandins and interferon-γ

Mononuclear inflammatory cells (MC) isolated from the livers and spleens of mice with chronic graft-vs-host disease (CGVHD) to minor histocompatibility antigens (B10.D2 → BALB/c) show defective proliferation when stimulated with Con A and LPS. In turn, both CGVHD liver and spleen cells suppress the proliferation of mitogen-stimulated normal spleen cells in a genetically unrestricted manner. The suppressor activity of CGVHD spleen cells is mediated by plastic non-adherent null (natural suppressor) cells and involves a soluble suppressor factor(s). In contrast, the suppressor activity of CGVHD liver cells is mediated by macrophages (Mφ). In the current studies we show that the suppressor activity of CGVHD liver cells is also mediated by soluble factors and compare the roles of prostaglandins and interferon (IFN)-γ in mediating defective proliferation and suppressor activities of CGVHD liver and spleen MC. Monoclonal antibody to IFN-γ partially reversed the defective mitogen-stimulated proliferation of CGVHD spleen MC but had no effect on proliferative response of CGVHD liver MC. Indomethacin did not alter the low proliferative response of either CGVHD liver or spleen MC. Anti-IFN-γ inhibited the ability of CGVHD spleen cells to suppress proliferation of Con A and LPS-stimulated B10.D2 spleen cells. In contrast, anti-IFN-γ resulted in a small decrease in the ability of liver MC to suppress Con A (but not LPS)-stimulated cell proliferation. Indomethacin decreased the ability of both CGVHD liver and spleen cells to suppress Con A-stimulated proliferation but had inconsistent effects on LPS-stimulated proliferation. These results show that IFN-γ and prostaglandins partially mediate the suppressor activity of CGVHD spleen MC. The suppressor activity of CGVHD liver MC also involves prostaglandins but is relatively independent of IFN-γ.

Mechanisms of complete Freund's adjuvant protection against diabetes in BB rats: induction of non-specific suppressor cells.

We have previously reported that a single injection of complete Freund's adjuvant (CFA) can prevent diabetes appearance in diabetes-prone (DP) BB rats. In this study, we investigated further the mechanism of CFA-induced protection from diabetes. We found that adoptive transfer of splenic cells from CFA-treated DP rats into young DP rats protected the latter from diabetes development. This suggested that CFA-induced protection from diabetes resulted from activation of regulatory (suppressor) cells. Cell mixing experiments in vitro indicated that CFA activated splenic cells with antigen-nonspecific suppressor activity (suppression of lymphoproliferative responses to lipopolysaccharide and to allogeneic splenic cells). Fractionation of splenic cells on Percoll revealed that the suppressor activity resided in low density cells relatively depleted of T-cells, B-cells, macrophages and NK cells. These results suggest that non-specific (natural) suppressor cells in CFA-treated BB rats may be responsible for suppressing autoimmune responses and preventing insulitis and diabetes development.

Studies of Mammalian Implantation In Vitro. (I): Production of Laminin from Human Decidual Cell Line and Its Effects on Murine Trophoblastic Outgrowth.

TTK-1 cell line from human first trimester decidual tissue carried the surface markers such as HNK-1 (Leu 7) and HLA-DR. This cell line was strongly suspected of being human natural suppressor cell line of bone marrow or lymphoid tissue origin. In addition, the cells were found to express laminin on its cell surface, as examined by indirect immunofluorescence (IIF) test, and also secrete it into culture supernates as examined by enzyme linked immunoassay (EIA) and immunostaining. The addition of the supernates from this cell line into the murine embryo culture induced the trophoblastic outgrowth of embryos in vitro in the same manner as murine EHS sarcoma derived laminin did. This phenomenon was considered to be due to laminin in the UK-1 supernates because the effect was completely blocked by the addition of anti-laminin antibodies into the embryo culture.

Proliferation of natural suppressor cells in long-term cultures of spleen cells from normal adult mice.

Natural suppressor cells were induced by culturing spleen cells from normal adult mice for 2 to 3 wk. The suppressor cells were large in size, nonadherent and nonspecifically suppressed the plaque-forming cells response of fresh spleen cells to SRBC in vitro. The suppressive activity of the cells was not affected by treatment with indomethacin or anti-Thy-1, anti-Ig, anti-Ia, or anti-asialoGM1 plus complement. Phenotype analysis by FACS showed that Thy-1, L3T4, Ly-2, CD3-epsilon, TCR-alpha beta, Ig, B220, Ia, and asialoGM1 Ag were all absent in the suppressor cells, although they were wheat germ agglutinin receptor positive. The suppressor cells did not demonstrate cytotoxicity against either YAC-1 or P-815 cells. Enriched large cell populations from fresh normal spleens expressed the same phenotypes and also exhibited the suppressive activity. These findings suggest that a minor population of natural suppressor cells exist in the normal adult mouse spleen and they proliferate during the in vitro culture of spleen cells.

Identification of a factor(s) from cloned murine natural suppressor cells that inhibits IL-2 secretion during antigen-driven T cell activation

Crude supernatants were obtained from cloned murine natural suppressor (NS) cells activated in vitro with phorbol-myristate-acetate (PMA) and calcium ionophore (A23187). Supernatants suppressed IL-2 production in the mixed leukocyte reaction (MLR) with BALB/c spleen cells, but no reduction was observed in the response to PHA, Con A, or anti-CD3 antibody. Suppressive activity was partially purified by DEAE ion exchange chromatography, and inhibited the antigenpresenting function of the macrophage line 1G18-LA in an assay system with the ovalbumin-specific T cell hybridoma, 3DO-18.3. In addition, the antigen-presenting function of the A20 B cell line was inhibited in an assay with a sperm whale myoglobin (SpWMb)-specific T cell hybridoma (8.2.1d.H1a). Results with blocking antibodies suggest that this factor appears to be a unique cytokine.

CD3+ T Cells in Severe Combined Immunodeficiency (SCID) Mice. V. Allogeneic T Cells Engrafted into SCID Mice Do Not Induce Graft‐Versus‐Host Disease in spite of the Absence of Host Veto and Natural Suppressor Cell Activity

Intravenous injection of 10(6) to 10(7) non-fractionated spleen cells (SC) from C57BL/6 (B6, H-2b) mice into completely allogeneic, immunodeficient H-2d severe combined immuno deficiency (scid) mice leads to engraftment of allogeneic donor T cells. Mice analysed in the tenth week posttransfer had engrafted donor-type CD4+ and CD8+ T cells in the spleens but showed no clinical evidence of graft-versus-host disease (GVHD). Transfer of allogeneic T cells engrafted in scid recipients did not induce GVHD upon i.v. injection into secondary scid recipients and lead in most recipients to engraftment of a pure CD4+ T-cell population. Experiments were carried out to investigate the reason(s) for the lack of GVHD in recipient scid mice, i.e. the presence of allotolerance in the engrafted donor T cells. Scid spleen cells (SC) efficiently stimulated alloreactive responses of B6 T cells: scid SC stimulated H-2d-specific cytotoxic responses in a B6 anti-scid mixed lymphocyte culture in vitro, and scid SC injected i.v. into B6 mice efficiently primed splenic cytotoxic lymphocyte precursors against H-2d alloantigens. Moreover when assayed in vitro, no veto activity or natural suppressor activity was detectable in scid SC. These data demonstrate that tolerizing mechanisms currently believed to operate in vivo can not explain the fact that allogeneic T cells injected i.v. into immunodeficient scid mice become tolerized against host-type alloantigens. Our results are discussed in the light of clinical experience of allogeneic T-cell transfer in scid infants.

SUPPRESSION OF MIXED LYMPHOCYTE REACTIVITY BY MURINE BONE-MARROW-DERIVED SUPPRESSOR FACTOR-INHIBITION OF PROLIFERATION DUE TO A DEFICIT IN IL-2 PRODUCTION

Murine bone marrow natural suppressor cells constitutively produce a soluble factor, BDSF, that suppresses murine Ab responses in vitro. BDSF is recovered in supernatants of cultured BM cells and resides in an MW range between 1-10 kD. We now demonstrate that BDSF suppresses proliferation in the mixed lymphocyte reaction (MLR). In order to elucidate the potential mechanism of action, BDSF was tested for the ability to interfere with IL-2-mediated T cell proliferation. BDSF was not able to inhibit IL-2-driven proliferation of the cell lines CTLL-2 and HT-2. However, examination of MLR culture supernatants revealed that suppression of proliferation by BDSF was associated with decreased levels of IL-2 production. Furthermore, responsiveness of BDSF-treated cultures could be reconstituted by adding exogenous IL-2, at the time of plating. The results suggest that hyporesponsiveness in the presence of BDSF is due to a deficit in IL-2 production, preventing expansion of Ag-stimulated cells.

SEPARATION OF ALLOSTIMULATORY AND NATURAL SUPPRESSOR/STEM CELL FUNCTIONS OF MURINE BONE MARROW—IMPLICATIONS FOR BONE MARROW TRANSPLANTATION

Natural suppressor (NS) activity is defined by the ability of a null cell population to inhibit antigenic and mitogenic activation of lymphoid cells. We had previously demonstrated that murine and human bone marrow was a rich source of NS activity and could be enriched in a population of large, low-density cells after counterflow centrifugal elutriation (CCE). In this report we confirm previous findings in the mixed lymphocyte reaction (MLR) that NS activity is an endogenous function of BM and is not present in peripheral immune tissues. Furthermore, suppression by BM NS cells in the MLR is not MHC restricted, which distinguishes NS activity from veto activity also associated with BM. To enrich for NS activity, BM cells were separated into 3 fractions according to size and density by CCE. Fraction (F) 1 contained small, high-density cells; F2 contained cells of intermediate size and density; and F3 comprised large, low-density cells. Addition of CCE-enriched fractions to the MLR revealed potent NS activity associated with F3, consistent with our previous findings in the Ab response. CCE-enriched fractions of BM cells were also used as stimulators in an MLR to determine which fraction, if any, could support alloantigen-induced proliferation. The use of unfractionated C57Bl/6 (H-2b) BM cells as stimulators for BALB/c (H-2d) splenic responders resulted in little detectable proliferation as compared with that induced by C57Bl/6 splenic stimulators. However, when elutriated fractions were used as stimulators, F1 BM cells induced a significant proliferative response, albeit to a lesser magnitude than spleen cells. In order to determine which fraction contained hematopoietic progenitors, CCE-separated cells were assayed for granulocyte/macrophage colony formation (CFU-GM). F3 BM was enriched five-fold for CFU-GM progenitors as compared with unfractionated BM, while progenitors were virtually absent in F1 and F2 BM. The results suggest that CCE may represent an effective way of removing potential alloantigen-presenting cells (F1) from BM, while retaining hematopoietic progenitors and NS activity (F3). The implications of the findings as they relate to allogeneic BM transplantation are discussed.

Regulation of thymocyte proliferation by α β TcR + CD3 + CD4 − CD8 − cloned natural suppressor (NS) cells

Cloned α β TcR + CD3 + CD4 − CD8 − T cells, with natural suppressor (NS) activity, were cocultured with thymocytes in the presence or absence of mitogen or cytokines. Whereas thymocytes show a minimal response to phytohemagglutinin (PHA), IL-2, or IL-4 alone, they proliferate vigorously when cocultured with irradiated cloned NS cells in the presence of PHA or IL-2 or IL-4, but not with IL-1, IL-3, IL-6, IL-7, IFN-γ, or GM-CSF. Among a total of 11 NS cell clones, derived from the spleen or thymus, only one clone (NR-1) did not induce thymocyte activation in synergy with PHA. This costimulation is most likely mediated by soluble factor(s), since supernatants, obtained from NS cells activated with phorbol ester (PMA) and calcymicin (A23187) or with solid phase anti-CD3 mAb, enhance thymocyte DNA synthesis in the presence of a mixture of PHA, IL-2, and IL-4. The latter factor does not appear to be a previously described lymphokine, since PMA- and A23187-activated NS cells secrete IL-3, TGF-β, IFN-γ, GM-CSF, and TNF-α, but not IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, nor IL-10. None of the factors, identified in the NS cell supernatants, was able to stimulate thymocyte DNA synthesis.This study shows that, in addition to their previously reported suppressor function, cloned NS cells can exert immunostimulatory activities by virtue of a soluble mediator.