An in vitro system for the generation of suppressor cells and the requirement for B cells in their induction

An in vitro method for the generation of effector suppressor cells (Ts3) was developed. By utilizing this protocol, it was possible to investigate both the cellular and genetic requirements for suppressor cell induction. It was determined that populations containing Ts3 cells can be induced after a 4-day culture of spleen cells and antigen. These Ts3 cells are similar to Ts3 cells generated by in vivo immunization. Both populations are I-J+, bind NP hapten, bind NP hapten, bear receptors which share NPb idiotypic determinants with anti-NP antibodies, function during the effector phase of the immune response, and require activation with Ts2 cells. Generation of Ts3-containing populations required both nylon wool-nonadherent T cells and a nylon-adherent, B cell-enriched population from an Igh-identical donor. T cells cultured with antigen alone or with syngeneic macrophages and antigen did not develop suppressive activity. Lytic treatment of the nylon-adherent population with a B cell-specific monoclonal antibody (J11d) removed the ability to generate suppressor cells. These results imply that the induction of suppressor T cells requires B lymphocytes, and that this induction process is dependent on Igh-linked gene products.     

Further characterization of suppressor lymphocytes induced by fetal calf serum in murine lymphoid cell cultures: comparison with in vitro-generated cytotoxic lymphocytes.

Nonspecific suppressor cell (SPC) activity has been induced in vitro by preculturing splenocytes from normal mice in the presence of fetal calf serum (FCS) for 3 days or more. In adoptive transfer experiments in vivo, these precultured SPC were shown to reduce the humoral response of mice to SRBC and the cell-mediated cytotoxic (CMC) response to allogeneic tumor cells. In mixing experiments in vitro, using freshly explanted splenocytes, the precultured splenocytes abrogated the generation of specific cytotoxic lymphocytes (CL) in primary mixed lymphocyte-tumor cell cultures (MLTC). By contrast, secondary cytotoxic response was only marginally affected. Supernatants of precultured cells were also inhibitory, although to a lesser degree than whole cells. The induction of suppressive activity was abolished by addition of mitogenic amounts of concanavalin A to the preculturing medium.By the use of cell fractionation techniques it was found that both specific CL and nonspecific SPC lack an Fc receptor, do not adhere to nylon wool, and cannot be separated from each other by density sedimentation on a discontinuous BSA gradient. However, precursors of SPC and CL differed in their susceptibility to cyclophosphamide, hydrocortisone, and irradiation. The data presented does not exclude the possibility that suppressive activity exerted by FCS-induced SPC is mediated through a cytotoxic effect.     

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.     

Regulation of the In Vitro Secondary Cell-Mediated Cytotoxic Response against Syngeneic FBL-3 Leukemia by Macrophages

Depletion of macrophages from immune spleen cells by treatment with carbonyl iron and magnet or by in vivo treatment with carrageenan enhanced the in vitro secondary cell-mediated cytotoxic response against a syngeneic Friend virus-induced leukemia, FBL-3 cells of C57BL/6 mice. However, further depletion of macrophages by passing the carbonyl iron-treated immune spleen cells through a nylon wool column abrogated the cytotoxic response. The addition of splenic macrophage-enriched preparations from either FBL-3-immune or normal mice suppressed the cytotoxic response of immune spleen cells treated with carbonyl iron and magnet. This suppressive effect of splenic macrophages presented a marked contrast with the enhancing effect of normal peritoneal macrophages on the same cell-mediated cytotoxic response, indicating regulation of the generation of killer T cells against a syngeneic tumor by functionally distinct macrophages. The suppressed cell-mediated cytotoxic response against FBL-3 cells by immune spleen cells was augmented by the addition of indomethacin to the culture medium, and this augmentation with indomethacin was greatly decreased by depletion of phagocytic cells from the immune spleen by treatment with carbonyl iron and magnet. The mechanisms of regulation of the cell-mediated cytotoxic response with soluble factors released from macrophages are discussed.     

Suppressor T cells induced by soluble immune complexes can adoptively transfer inhibition of cytophilic antibody receptors on macrophages.

Immune complexes (soluble antigens of L1210 and antibody to L1210) when given to allogeneic C3H mice generated suppressor cells that inhibited receptors for cytophilic antibody on macrophages. Thymocytes or nylon-nonadherent splenic T cells (4 × 10⁷) from immune-complex-treated mice transferred this suppressive activity when injected into normal syngeneic mice. Maximal suppression of macrophages occurred 4 to 6 days after transfer. In contrast, even 5 × 10⁷ nylon-adherent, non-T spleen cells from immune-complex-treated (“suppressed”) mice failed to induce macrophage suppression in the syngeneic recipients. When T-cell-depleted “B” mice were used as recipients, neither thymocytes nor splenic T cells from suppressed mice were able to transfer suppressive activity. However, the admixture of 2 × 10⁷ normal syngeneic thymocytes with 4 × 10⁷ thymocytes from suppressed mice restored the latter's ability to elicit suppression of macrophages in T-cell-deprived recipients. Peritoneal monocytes from recipients of suppressor thymocytes (to L1210) could not attach cytophilic antibody to L1210 but could attach cytophilic antibody to EL-4 and sheep erythrocytes. Thus, suppressor T cells induced by immune complexes can transfer immunologically specific macrophage suppression (inhibition of cytophilic antibody receptors) to syngeneic recipients. The suppressor cells required the cooperation of normal T cells, suggesting either recruitment of suppressor cells from, or a helper effect by, the normal T cells, in order to produce their effect.     

Specificities of killing by cytotoxic lymphocytes generated in vivo and in vitro to syngeneic SV40 transformed cells.

Cell-mediated immunity to SV40-transformed C3H and C3H-SW cell lines was measured by using both 51Cr and 125IUdR release assays. Killing by cytotoxic cells generated on in vitro sensitization of immune spleen cells with syngeneic SV40 cells by either assay is specific for syngeneic SV40 transformants. Cytolysis mediated by in vitro sensitized cells is ablated by treatment of the effector cells with anti-theta serum and complement. Intraperitoneal immunization with syngeneic SV40 cells yields two distinct killer-cell populations in the peritoneal exudate when assayed by 125IUdR release. The first, nylon wool nonadherent and sensitive to anti-theta and complement, is indistinguishable from the killers generated in vitro. The second population, present in larger numbers and more efficient on a per-cell basis in killing of SV40 targets than the first, is nylon adherent and is not removed by treatment with anti-theta and complement. This second population will kill any SV40 transformed target, whether syngeneic or allogeneic. The possible roles of T cell and non-T cell effectors in rejection of syngeneic SV40 tumors are discussed.     

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.     

C-Reactive protein (CRP)-mediated inhibition of the induction of in vitro antibody formation. I. T-cell dependence of the inhibition.

Purified human C-reactive protein (CRP) inhibited the in vitro anti-hapten antibody plaque-forming cells (PFC) response of both carrier keyhole limpet hemocyanin (KLH)-primed and unimmunized Balb/c spleen cells to TNP-KLH. The inhibitory effect was neutralized by the CRP-substrate, C-polysaccharide. The response to the T-independent antigens, TNP-T4 and DNP-lys-Ficoll, was not inhibited by CRP. A cell population that was suppressive for the in vitro PFC response was generated by incubating normal spleen cells with CRP. These cells suppressed the PFC response of syngeneic KLH-primed cells to TNP-KLH in proportion to the number of added lymphoid cells with bound CRP. Selective depletion of B cells, T cells or macrophages before incubation with CRP revealed that T cells were required for the induction of suppressive cells. Treatment of spleen cells after incubation with CRP, with T cell-specific antisera and C abolished suppressor-cell activity. Mitomycin-C treatment of the CRP-binding cells did not alter their suppressive activity. These results indicated that CRP mediates suppression of antibody induction to T-dependent antigens by interacting with T cells and generating a suppressive T-cell population.     

Specific inhibition of hybrid resistance in F1 hybrid mice pretreated with parent strain spleen cells. I. Induction of a nylon-adherent, Thy-1+Lyt-1+2- suppressor cell

Hybrid resistance, which is observed in certain strain combinations when parent-strain bone marrow cells are grafted into lethally irradiated F1 hybrids, can be specifically overcome by the i.v. injection, 1 wk before the graft, of spleen cells syngeneic with the bone marrow graft. This phenomenon is due to a suppressor mechanism, induced in the spleen of the F1 hybrid by the injection of parent-strain spleen cells and mediated by a nylon-adherent Thy-1+Lyt-1+2- cell population of hybrid origin, because hybrid resistance can be inhibited by the transfer into a normal B6D2F1 of nylon-adherent Thy-1+Lyt-1+2- spleen cells from B6D2F1 mice pretreated with B6 spleen cells 1 wk earlier (B6-pretreated B6D2F1); spleen cells from B6-pretreated B6D2F1 mice not depleted of their nylon-adherent subpopulation cannot restore hybrid resistance when they are injected into a B6D2F1 rendered nonresistant by split-dose irradiation; and spleen cells from normal B6D2F1 mice cannot restore hybrid resistance when they are injected into B6-pretreated B6D2F1 hybrids. The suppressor cells specifically inhibit resistance against bone marrow cells syngeneic with the spleen cells used for pretreatment, because transfer of nylon-adherent B6-pretreated B6D2F1 spleen cells into a normal B6D2F1 does not enhance syngeneic B6D2F1 or parent-strain D2 bone marrow growth, and when injected into normal B6D2F1 hybrids, nylon-adherent spleen cells from B6D2F1 mice pretreated with D2 spleen cells 1 wk earlier (D2-pretreated B6D2F1) are not able to transfer the inhibition of hybrid resistance against B6 bone marrow cells. Moreover, the activity of the suppressor cells depends on the genetic environment of the hybrid host mice, because nylon-adherent B6-pretreated B6D2F1 spleen cells injected into normal B6C3F1 hybrids do not transfer an inhibition of hybrid resistance, and when injected into B6C3F1 hosts previously rendered nonresistant by split-dose irradiation, spleen cells from B6-pretreated B6D2F1 mice can, in contrast, transfer hybrid resistance.     

Regulatory mechanisms in cytotoxic T lymphocyte development. II. Dissociation of in vitro cytotoxic T-lymphocyte and suppressor T-cell activities with L-ornithine

L-ornithine was found to differentially affect the induction of allospecific cytotoxic T lymphocytes (CTL) and suppressor T cells (Ts). At a concentration of 10 mM, ornithine inhibited the development of CTL in a mixed-leukocyte culture (MLC). This same population of cells suppressed the generation of CTL when irradiated and cocultured with fresh syngeneic lymphocytes and alloantigen. Suppression was mediated by Lyt-1-2+ cells and was antigen specific. Suppression was abrogated when IL-2 (10 U/ml) was added to the cocultures, but could not be reversed by increasing the antigen dose. Ornithine was not toxic to CTL precursors but rather arrested their development. Cells from MLC plus ornithine developed CTL activity within 2 days of transfer to secondary cultures in the absence of ornithine. Development of CTL effector cells (CTLe) was augmented by but did not require exogenous IL-2. Generation of CTLe from the MLC plus ornithine population was radiation sensitive and could be inhibited by reexposure to ornithine, even in the presence of IL-2. Thus, Lyt-1-2+ T cells allostimulated in vitro in MLC plus ornithine and lacking CTL activity convey radiation-resistant, antigen-specific suppression.     

The suppressive effect of a supernate from concanavalin A-activated human lymphocytes: effects of concanavalin A-activated lymphocytes and their supernates on cytotoxic and mixed lymphocyte reactions.

The effects of Concanavalin A-treated human peripheral blood lymphocytes and their supernatants were evaluated on the MLC reaction and on the generation of cytotoxic lymphocytes assessed by cell-mediated lympholysis (CML). Experiments were performed with both allogeneic and xenogenic sensitization. It was found that Con A-activated cells suppressed the MLC and CML reactions in allogeneic and xenogeneic systems. On the other hand, the SIRS-like supernate was able to suppress the MLC reaction and blastogenesis, but had no suppressive effect on the generation of cytotoxic lymphocytes. We found no difference in the magnitude of suppression, whether or not Con A-activated lymphocytes were syngeneic to the responder cells. This finding suggests that there is no requirement for allogeneic restriction in the interaction between suppressor and suppressed cells, and demonstrates a soluble human suppressor substance capable of suppressing some cell-mediated reactions.     

Cellular requirements for the generation of primary cell-mediated lympholysis responses to Qa-1 antigens

Mixed leukocyte cultures (MLC) between NZB responder spleen cells and Qa-1-disparate stimulator spleen cells were employed to determine the cellular requirements for the generation of primary anti-Qa-1 cell-mediated lympholysis (CML) responses. Although primary anti-Qa-1 cytotoxic lymphocytes (CTL) were generated during H-2-homologous stimulation, anti-Qa-1 CTL were not detectable from MLC in which the stimulators were H-2 allogeneic. Anti-Qa-1 CTL also were not generated from MLC in which the stimulators were semiallogeneic. Thus, H-2 identity between responder and stimulator cells was not sufficient to permit the generation of primary anti-Qa-1 CTL when H-2 disparity was also present. The capacity for H-2 disparity to prevent anti-Qa-1 CML responses was further demonstrated in MLC containing both H-2-allogeneic and H-2-homologous stimulator cells. Therefore, in subsequent studies we employed NZB responders and H-2-homologous, Qa-1-disparate stimulators. When various subpopulations of stimulator cells were studied for their ability to induce anti-Qa-1 CTL, nylon wool-adherent cells were found to be potent stimulators, but nylon wool-nonadherent cells were not. Furthermore, depletion of macrophages from the stimulator population abrogated the generation of anti-Qa-1 CML responses, despite the presence of responder macrophages in the culture. In contrast, all fractionated subpopulations stimulated anti-H-2 CML responses. When macrophage-enriched cells were used as stimulators, anti-Qa-1 CTL could be generated with approximately 80-fold fewer stimulator cells than when unfractionated splenocytes were used as stimulators. These findings indicated that stimulator macrophages were essential for the generation of primary anti-Qa-1 CTL. Direct evidence for macrophage expression of Qa-1-antigens was obtained by using a Qa-1b-specific CTL clone. These studies provide i) the first evidence for Qa-1 expression on macrophages, ii) a basis for comparison of the cellular interactions necessary to generate CTL against H-2K/D-encoded vs Qa-1-encoded class 1 antigens, and iii) a model for investigating the mechanisms responsible for the immunodominance of H-2K/D alloantigens.     

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.     

Mycobacterium leprae antigen-induced suppression of T cell proliferation in vitro

The extent to which M. leprae and its products induced suppression of T lymphocyte proliferation in vitro was evaluated. M. leprae antigens suppressed T cell proliferation in response to mitogens and antigens in both lepromatous and tuberculoid patients, as well as controls never exposed to M. leprae or M. leprae endemic areas. Both soluble and particulate fractions of M. leprae were found to suppress proliferation in a dose-dependent manner. The extent of suppression was inversely related to the proliferative response of the donors mononuclear cells to M. leprae. Evidence indicates that M. leprae contains both stimulatory and suppressive molecules for T cells. One such suppressive antigen, Lipoarabinomannan (LAM)-B of M. leprae, also suppressed the proliferative response of tuberculoid patients. Suppression was also observed with the LAM-B of M. tuberculosis. The suppressive effects observed were not due to the toxicity of the antigen. Some of the suppressive activity was mediated by T8+ suppressor cells and was expressed in both lepromatous and tuberculoid patients. We suggest that previous sensitization to M. leprae and other cross-reactive mycobacterial antigens determines the sensitivity of T cells to the suppressive effects of M. leprae antigens.     

Suppressor cell activity in tumor-bearing mice. I. Dualistic inhibition by suppressor T lymphocytes and macrophages.

Spleen cells from mice bearing methylcholanthrene-induced fibrosarcomas impaired mitogen responses of normal syngeneic lymphocytes. Nylon wool column and other depletion techniques were utilized to characterize the cellular source of suppressive activity in tumor-bearing host (TBH) spleens. Evidence is presented for two distinct suppressor cell systems operating in the spleens, but not lymph nodes, of BALB/c mice bearing transplanted tumors. Spleens from TBH were shown to have greatly increased numbers of macrophages over their normal counterparts. TBH macrophages were observed to have suppressive activity at low in vitro concentrations. Anti-Thy 1 serum treatment of TBH macrophages abrogated low dose inhibition but not suppression due to high numbers of macrophages. No functional difference was detected between anti-Thy 1 serum-treated TBH and normal splenic macrophages. In a macrophage-depleted culture system, mildly nylon wool adherent, anti-Thy 1 serum, and hydrocortisone succinate-sensitive suppressor cells could be detected. Soluble supernatant products of TBH spleen and thymus cells were also found to inhibit in vitro mitogen responses, whereas TBH macrophages and lymph node cells demonstrated no soluble suppressive activity. The major source of soluble inhibitor of DNA synthesis (IDS) seems to be an anti-Thy 1 serum, hydrocortisone-sensitive population.     

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.     

Studies on the specificity of in vitro induced lymphocytotoxicity to SV40-transformed fibroblasts.

Cytotoxic effector lymphocytes were induced by in vitro immunization of lymph node and spleen cells from AKR-mice (H-2k) and from BALB/c-mice (H-2d) to syngeneic SV40-transformed fibroblasts. The T cell-dependent cytotoxicity was specific for target cells expressing the same H2-specificity as the immunizing cells. Nontransformed fibroblasts as stimulator cells did not induce efficient cytotoxicity to transformed or nontransformed target cells. Incubation with phytohemagglutinin during the sensitization period modified the specificity of the T cell-mediated lysis of syngeneic SV40-transformed fibroblasts: allogeneic as well as syngeneic target cells were destroyed by these effector cells. However, the polyclonal stimulant activates preferentially cytotoxicity to H2-matched target cells. The in vitro generation of cytotoxic effector cells was restricted to living SV40-transformed fibroblasts as immunizing cells; it was not possible to immunize lymphocytes in the presence of membrane proteins prepared from the SV40-transformed cells. The cytotoxicity of the in vitro immunized lymphocytes was inhibited by incubation with membrane protein preparations from syngeneic or allogeneic SV40-transformed fibroblasts.     

Regulation of a cytotoxic response toward alloantigens by T-amplifier and non-T-suppressor cells.

A system is described by which it is possible to generate in vitro non-specific T-amplifier and non-T-suppressor cells without accompanying formation of CTL. Such cells could be separated and tested for their capacity to regulate generation of CTL towards H-2 alloantigen. The experimental setup was as follows: M-locus or syngeneically activated spleen cells were fractionated on BSA-gradients and added to fresh, nylon wool purified (T) precursors. These cultures were stimulated during a second stage with H-2 alloantigen and lytic activities monitored with the chromium release assay. It was found that M-locus activated cells contained two separable subpopulations: 1) an anti-Thy 1 sensitive, non-adherent amplifier cell of intermediate density and 2) a blast cell which was insensitive to anti-Thy 1 as well as RaMIg treatment (i.e. a “null” cell). This cell was adherent, but non-phagocytosing and had strongly suppressive effects on the generation of CTL. It also suppressed—at least partially—the activity of fully differentiated CTL. In syngeneically stimulated cultures strongly enhancing T amplifier cells with blast cell characteristics were found.     

Characterization of suppressor cells in mice bearing syngeneic mastocytoma.

Suppressor cells from syngeneic P815 mastocytoma-bearing DBA/2 mice that inhibit in vitro generation of specific anti-tumor cytotoxicity were characterized. Suppressive activity was almost completely eliminated by treating suppressive spleen cells with anti-theta serum and complement. Treatment with anti-mouse lg serum and complement or with carbonyl iron did not affect their suppressive activity. When suppressive thymocytes from P815 tumor-bearing DBA/2 mice were tested for their capacity to inhibit the generation of cytotoxicity against L1210 cells, a leukemia line in DBa/2 mice, they did not affect the activity, indicating that the supressor cells in the thymocytes of P815 tumor-bearing mice are specific to the tumor. When Ficoll-Hypaque density cell separation was carried out with cytotoxic spleen cells and suppressive spleen cells from 815 tumor-bearing mice, the dense fraction was enriched for kiler cells whereas the suppressive activitty was mainly recovered in the light fraction. Therefore, killer cells and suppressor cells in P815 tumor-bearing mice are thought to be distinct populations.     

Asialo-GM1+ natural killer cells directly suppress antibody-producing B cells

Natural killer (NK) cells were tested for their ability to suppress antigen-induced antibody responses in vitro. Asialo-GM1+ (ASGM1+) cells were prepared from nylon-wool-nonadherent spleen cells obtained from normal mice. After depletion of Ig+, L3T4+ and Lyt-2+ cells, the ASGM1+-enriched cell population had high NK activity which was abrogated by treatment with anti-ASGM1 and C'. This NK-enriched ASGM1+ cell fraction significantly suppressed the generation of antibody-producing cells when added to in vitro immunization cultures of primed spleen cells. Treatment of the NK-enriched cell population with anti-ASGM1 and C' abrogated the ability of these cells to suppress antibody responses. In vitro antibody production by purified B cells was also suppressed in the presence of the NK-enriched cell population, although the kinetics of the suppression differed from that observed with unfractionated spleen cells. In addition, the NK-enriched cell population suppressed the proliferation of the B cell line WEHI-279.1. Suppression of WEHI-279.1 cells was abrogated when the NK-enriched cell population was treated with anti-ASGM1 and C'. These results suggest that normal NK cells suppress the generation of antibody-producing B cells and that this occurs, at least in part, through a direct regulation of the B cell.