Normal human bone marrow suppressor cells and in liver cirrhosis

Suppressor properties of bone marrow cells were studied in healthy donors and patients with hepatocirrhosis using the technique registrating the activity of bone marrow B-suppressors by the inhibition of xenogenic target cell proliferation. The activity of bone marrow suppressor cells in patients with various types of hepatocirrhosis was reduced as compared to healthy subjects. In addition, the in vitro spontaneous proliferation level of bone marrow cells in hepatocirrhosis was considerably higher than that of healthy donors. This fact can be possibly attributed to the decline in the number of bone marrow B-suppressors or inhibition of their functional activity in hepatocirrhosis. Peripheral blood lymphocytes of these patients, like the lymphocytes of healthy donors, showed practically no suppressive effect in vitro.     

Regulation of autoimmunity in normal and rheumatoid individuals by bone marrow-derived natural suppressor cells and their suppressor factor: BDSF

Natural suppressor activity is defined as the ability of unstimulated effector cells to suppress responses of lymphoid cells to antigenic and mitogenic stimuli in an MHC unrestricted manner. We have described natural suppressor cells in bone marrow exerting such a function through a suppressor factor termed BDSF. In this report we demonstrate the ability of BDSF to regulate the in vitro EBV- and PWM-induced production of both IgM and IgM rheumatoid factor antibodies of lymphoid cells obtained from normal individuals and those with rheumatoid arthritis. BDSF therefore may play a role in the normal BM microenvironment suppressing primary IgM antibody responses. Since autoantibody responses are similarly suppressed, functional alteration or lack of BDSF may be responsible for the emergence of autoantibody-producing cells in bone marrow during certain autoimmune states.     

Bone marrow analysis of the myelodysplastic syndromes: histological and immunohistochemical features related to the evolution of overt leukemia

Bone marrow trephines from 31 patients with an initial diagnosis of myelodysplastic syndromes (MDS) were examined and analyzed histologically and immunohistochemically. In those cases terminating in overt leukemia (6/31, 19%), the number of bone marrow mast cells was significantly reduced, compared with those which did not evolve to overt leukemia. The bone marrow lymphoid cells that may participate in immunosurveillance against the proliferation of blast cells were also significantly reduced in cases terminating in overt leukemia. However, S-100 protein-positive cells, which include histiocytes and suppressor T-cells, were increased in cases terminating in overt leukemia. The results indicated that examination of the bone marrow to determine the proportions of mast cells and lymphoid cells which may be involved in host defense systems may be useful in predicting the evolution to overt leukemia in MDS. In the present series, patients with a hypocellular marrow (5/31, 16%) did not progress to overt leukemia and had a significantly lower bone marrow reticulin content, a significantly lower megakaryocyte count, a relatively higher mast cell count and a significantly higher lymphoid cell count than those with a normocellular or hypercellular marrow. These findings may reflect the initial features of MDS or, possibly, that hypocellular MDS is an independent entity with a low potential for blastic proliferation.     

The effect of an antigenic stimulation on proliferative response in athymic mice

The proliferation activity of the main cellular categories of bone marrow after infusion of³H-thymidine was studied in nu/nu and +/+ 1-month- and 3-month-old BALB/c mice in comparison with lymphoid cells in the spleen and mesenteric lymph nodes. The stem cell defect in nu/nu mouse bone marrow is compensated by an, increased proliferation in myeloid series and in agranulocytes. The increase of proliferation activity among lymphoid cells in peripheral lymphoid organs was observed only in the 3-month-old mice with a delay in the nudes.     

A soluble factor produced by bone marrow natural suppressor cells blocks interleukin 2 production and activity

We previously reported that a population of Fc gamma-receptor+ (Fc gamma R+) suppressor cells present in normal unstimulated rabbit bone marrow inhibited the growth of autologous rapidly proliferating bone marrow cells devoid of Fc gamma R. It is now reported that the Fc gamma R+ bone marrow cells produced a soluble, nondialyzable suppressor factor(s) (SF) which blocked the proliferation of Fc gamma R- bone marrow cells. In addition, the Fc gamma R+ cells and SF significantly inhibited spleen cell proliferation in response to concanavalin A (Con A), phytohemagglutinin, and pokeweed mitogen. The bone marrow SF exhibited a dose-dependent suppression of the growth of IL-2-dependent T lymphocytes in the presence of IL-2. SF also completely blocked the production or release of IL-2 by Con A-stimulated T cells. Thus, these bone marrow natural suppressor cells produced a soluble factor, which regulated the growth of rapidly proliferating bone marrow cells and also regulated T cell reactivity by modulating IL-2 production and activity.     

Infection of bone marrow cells in vitro with FLV: Effects on stem cell proliferation,differentiation and leukemogenic capacity

Long-term cultures of proliferating hematopoietic stem cells derived from bone marrow permit the study of the interaction between murine leukemia virus (MuLV) infection and the proliferation and differentiation of stem cells. We have used this system to analyze the replication of different biological variants of MuLV in bone marrow cells; the effect of MuLV infection upon pluripotent stem cell (CFU-S) proliferation; and the effect of MuLV on differentiation of CFU-S along different hematopoietic pathways. Two MuLV variants were studied in detail: the Moloney strain of lymphatic leukemia virus (Mol-MuLV) and the erythroleukemic Friend virus complex (FLV) consisting of the lymphoid leukemia helper virus and the defective spleen focus-forming virus (SFFV). Mol-MuLV and its sarcoma virus pseudotype, MSV(Mol-MuLV), replicate efficiently in the bone marrow cultures; however, CFU-S are lost more readily than in uninfected cultures, and the cultures are soon represented by a majority population of mononuclear macrophages. On the other hand, infection with FLV produces a prolonged survival of the spleen colony-forming cells, CFU-S, and CFU-C (the committed granulocytic precursor cells). Production of erythroleukemogenic SFFV is maintained in these cultures for more than 40 weeks. No erythroblastic differentiation was observed in vitro, however, neither erythroblast precursor cells (CFU-E) nor hemoglobin-producing cells could be detected. This suggests that the target cell for FLV is an earlier precursor cell.     

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-gamma (rIFN-gamma) 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.     

The role of the thymus in the ontogeny of the immune system

The proper development of the organs of the immune system is dependent on at least three factors: (1) the development of anlagen with the capacity to trap antigens and support the proliferation of lymphoid and plasma cell precursors; (2) the production by the bone marrow of lymphoid and plasma cell precursors which seed in the lymphoid organs; and (3) the thymus, which seeds reactive cells to the lymphoid organs and produces a humoral factor stimulating antigen-triggered proliferation of primitive lymphoid and plasma cells. Studies on cell population changes in the lymph nodes following thymectomy in mice confirm earlier evidence that most cells produced in the thymus do not seed to the lymphoid organs, but die locally in the thymus.     

Mechanism of protection from graft-vs-host disease in murine mixed allogeneic chimeras. I. Development of a null cell population suppressive of cell-mediated lympholysis responses and derived from the syngeneic bone marrow component

Splenocyte populations from whole body-irradiated recipients of mixed T cell-depleted (TCD) syngeneic and allogeneic (complete H-2 disparity) bone marrow, or of TCD syngeneic marrow alone, contain cells with the ability to suppress the generation of cell-mediated lympholysis responses in vitro. This activity, which is present by 8 days after bone marrow transplantation and persists for several weeks, has been analyzed for possible veto-like or other specificity. Although reproducible patterns of suppression were observed, depending both on host strain and on the genetic combination of the response examined, the overall suppression in vitro most closely resembles that which has been ascribed to "natural suppressor" cells in other systems. The suppression appears to be mediated by a non-T cell, non-B cell, nonadherent, asialo GM1-negative population. Cold target inhibition and CTL activity of chimeric cells have been ruled out as factors contributing to the observed suppression. Significantly, in mixed chimeras, suppression was found to be mediated exclusively by cells which were syngeneic to the recipient in both recipient strains tested. The rapid development of this suppressive activity may explain the resistance to graft-vs-host disease conferred on whole body-irradiated mice by the addition of TCD syngeneic marrow to an allogeneic graft-vs-host disease-producing inoculum.     

Regulation of natural killer progenitors. Studies with a novel immunomodulator with distinct effects at the precursor level

NK cells originate from progenitors in the bone marrow and maturate independently of other lymphoid organs. NK cell maturation represents an important site for regulation of the level of NK activity and constitutes a potentially interesting target for therapeutic intervention. The effect of the immunomodulator Linomide (carboxamide-3-quinoline) on the regeneration of NK cells was studied in vivo after depletion of mature NK cells. Linomide significantly, although to various extent, accelerated the maturation of NK cells after specific depletion with antibodies to asialomonoganglioside, treatment with cyclophosphamide or lethal irradiation and syngeneic bone marrow grafting. Examination of the target cell spectrum lysed by spleen effector cells during Linomide treatment as well as studies of phenotype, clearly indicated that the effector cells studied were NK cells. Treatment of mice for 4 days with Linomide increased the frequency of bone marrow NK cell progenitors from 1/11,900 to 1/6,000 as judged by limiting dilution analysis. Direct addition of Linomide in vitro had no effect on cultures of mature NK cells from spleen, but had an additive effect to IL-2 on the generation of NK cells when added to bone marrow cultures. Our study indicates that different mechanisms exist for the regulation of progenitor and mature NK cells, and that the immunomodulator Linomide represent a potentially important tool for investigating the mechanisms governing NK cell maturation.     

Induction of murine lymphokine-activated killer cells by recombinant IL-7

The data demonstrate that IL-7, a cytokine that was originally identified, purified, and cloned based upon its ability to support the growth of pre-B cells in vitro, also induces proliferation and promotes the generation of lymphokine-activated killer (LAK) cell activity in populations of resting peripheral lymphoid cells. Although the kinetics of LAK induction by IL-7 (which peaked at days 6 to 8 of culture) was slower than that detected in cultures containing IL-2 (which peaked at day 4), IL-7 was significantly more effective at maintaining cytotoxic activity over longer periods of time, and greater viable cell recoveries, than was IL-2. A wide range of murine tumor target cells were found to be lysed in an MHC-unrestricted fashion by IL-7 induced LAK, but syngeneic Con A-induced lymphoblasts were not; nor were target cells from the human tumors K562 or Daudi lysed by IL-7 LAK. IL-7 LAK were induced in populations of lymphoid cells obtained from secondary lymphoid tissues (peripheral lymph nodes and spleen), but not from primary lymphoid tissues (thymus and bone marrow). LAK induced by IL-7 from unfractionated populations of lymphoid cells were completely eliminated by treatment with anti-CD8 or anti-Thy-1+C, and unaffected by treatment with anti-CD4, anti-asialo GM1 or anti-NK1.1+C. Interestingly, although no detectable CD4+ effector cells could be detected in populations of LAK generated from unfractionated populations of lymphoid cells stimulated by IL-7, they were found to be generated from populations of lymphoid cells from which CD8+ cells had been eliminated before being cultured in medium containing IL-7. These data suggest that CD4+ T cells do not normally give rise to IL-7-induced LAK unless they are first separated from CD8+ T cells. LAK induced by IL-7 appear to be distinct from LAK activity induced by IL-2 in that there is no detectable involvement of NK-like effector cells at either the precursor or effector cell stages.     

In vitro and in vivo analysis of bone marrow-derived CD3+, CD4-, CD8-, NK1.1+ cell lines

The development of methods of avoiding graft-versus-host disease (GVHD) while retaining the alloengraftment-promoting and anti-leukemic effects of allogeneic T cells is a major goal of research in bone marrow transplantation (BMT). We have recently obtained evidence suggesting that natural suppressor (NS) cells derived from T cell-depleted (TCD) syngeneic marrow can protect against GVHD while permitting alloengraftment. We have now attempted to enrich and then propagate NS cells in vitro, with the goal of obtaining an enhanced anti-GVHD effect by adoptive transfer in vivo. Two long-term cell lines were generated culturing BMC depleted of Mac1-positive cells and of Mac1-positive plus Thy1-positive cells in high concentrations of IL-2. Both cell lines showed anti-GVHD effects when administered along with a GVHD-producing inoculum, while permitting complete allogeneic reconstitution. A clone derived from Mac1-depleted BMC protected completely against a more chronic pattern of GVHD. These cell lines demonstrated suppressive activity in vitro, cytolytic activity against a broad range of natural killer (NK)-sensitive and NK-resistant targets, and a novel cell surface phenotype, with characteristics of both alpha beta-TcR-bearing T cells and of NK cells. In some respects, these cells resemble LAK cells and differ from fresh NS cells, and from the cloned NS cells derived from spleens of total lymphoid irradiation (TLI)-treated mice and neonatal mice. To our knowledge, this is the first detailed phenotypic analysis of cell lines with in vivo anti-GVHD activity. If applicability can be demonstrated in large animal models, the ability to use bone marrow as a source of such protective cell lines might also have potential utility in clinical BMT.     

A partial characterization of suppressor cells in the spleens of mice conditioned with fractionated total lymphoid irradiation (TLI)

Total lymphoid irradiation (TLI) is a highly effective modality for inducing immunosuppression and transplantation tolerance. The cellular basis for this immunosuppression is not clear, although T cells have been implicated. To study further the effect of TLI on the immune system, we have examined the B cells and suppressor cells in the spleens from TLI-conditioned mice. Our results indicate that after TLI, the spleen is rapidly repopulated with many large, immature cells. The probable source of these cells is the shielded bone marrow (BM). The B cells from TLI-conditioned mice are transiently immature and hyporesponsive in vitro to a T-independent antigen. Spleen cells from TLI-conditioned mice nonspecifically suppress the in vitro T-independent anti-TNP response of normal B cells. The suppressor cells lack both B and T cell markers and adhere to Sephadex G-10. The suppressor cells in spleens from TLI-treated mice bear a number of similarities to those present in normal BM. When normal BM cells were analyzed by indirect immunofluorescence for the presence of the Mac-1 antigen, two populations of suppressor cells could be identified: one was Mac-1+ and the other was Mac-1-. These data are consistent with the possibility that a subpopulation of the suppressor cells found in normal BM and in the spleens from TLI-conditioned mice are immature cells of the monocytic/granulocytic lineage.     

Migration of natural suppressor cells from bone marrow to peritoneal cavity by live BCG

Delayed-type hypersensitivity (DTH) responses were suppressed in mice inoculated with bone marrow cells from mice that had been injected with 10(8) colony-forming units (CFU) of live BCG. Upon analysis of this DTH-suppression by the use of a macrophage migration inhibition (MI) assay, the in vitro correlate of DTH, suppressor macrophages in the peritoneal cavity were found to play an important role in DTH suppression. However, neither suppression of DTH nor production of suppressor macrophages was observed in mice inoculated with bone marrow cells from mice that had been injected with methotrexate (MTX), a folic acid antagonist, and 10(8) CFU of live BCG. Moreover, suppressor cells against the MI activity of peritoneal exudate cells from BCG cell wall-immunized mice existed in bone marrow cells from normal mice, natural suppressor (NS) cells, and they were sensitive to MTX. In addition, these NS cells phagocytized carbonyl iron particles, were adherent to Sephadex G-10, and had Fc receptors, but they had no B or T cell markers, suggesting that these cells belonged to a macrophage compartment. From this evidence, we hypothesized that the origin of suppressor macrophages in the peritoneal cavity induced by live BCG injection was MTX-sensitive NS cells in bone marrow, and that these NS cells were stimulated by a small dose of live BCG trapped in bone marrow after i.v. injection of a high dose of live BCG and migrated from bone marrow to the peritoneal cavity.     

Genetic approach and phenotype-based complementation screening for identification of stroma cell-derived proteins involved in cell proliferation.

The functional capacities of stromal cell lines to support stem cell activity are heterogeneous and the mechanism of how they support bone marrow cultures remains unclear. Recently, we reported a strategy of functional analysis in which a genetic approach is combined with phenotype-based complementation screening to search for a novel secreted growth factor from mouse bone marrow stroma called ShIF that supported proliferation of bone marrow cells. To investigate the role of stromal cells in hemopoiesis, we extended this strategy to search for stroma-derived proteins that induce cell proliferation by establishing stroma-dependent Ba/F3 mutants of three stroma cell lines from two mouse tissues. Seven stroma-dependent Ba/F3 mutants were used as responder cells to identify cDNAs from stroma cell lines whose products supported proliferation not only to the mutant cells but also to hemopoietic progenitor cells in vitro.     

Suppression of cell-mediated immune responses after total lymphoid irradiation (TLI). I. Characterization of suppressor cells of the mixed lymphocyte reaction

Total lymphoid irradiation (TLI) was administered to (BALB/c X C57BL/6)F1 mice in eight daily doses of 200 rad (total 1600 rad). Spleen cells isolated from mice after treatment with TLI do not respond to alloantigens in vitro in a one-way mixed lymphocyte reaction (MLR), but normal reactivity recovers after approximately 2 mo. Radioresistant, antigen-nonspecific suppressor cells are documented in the spleens of TLI-treated mice immediately after radiotherapy, but suppressive capacity gradually disappears within 30 days. After TLI, the spleen is repopulated with large cells, the proportion of which is greatest at a time when theta-bearing cells are still depleted. Radioresistant suppression is mediated predominantly by the large cell subset and is thymus independent. Suppressor function can be abolished by lethal physicochemical procedures including formaldehyde fixation, multiple freeze-thawing, and heating to 56 degrees C, and it cannot be conferred by supernatants of TLI-suppressed MLR suspensions. Suppression cannot be overcome by adding various cell factors including T cell growth factor (TCGF) and lymphocyte-activating factor (LAF), nor is it affected by a prostaglandin inhibitor. Equally potent radioresistant suppressive activity is documented by co-culturing cells derived from other sources enriched in large, immature hematopoietic cells, including fetal liver cells and bone marrow cells obtained from normal and congenitally athymic mice. The presence of a large cell population and MLR suppressor function is also documented in the spleens of mice treated with single dose or fractionated doses of lethal whole body irradiation, followed by reconstitution with bone marrow cells obtained from normal mice. The data suggest that MLR suppressor cells, which are large, immature and predominantly radioresistant, can be induced after a short and well-tolerated TLI regimen.     

Failure of NZB spleen to respond to prethymic bone marrow suppressor cells.

Mouse bone marrow contains theta-negative lymphocytes that can suppress an in vitro plaque response by spleen cells primed in vivo with burro red blood cells (BRBC). These bone marrow cells are radiosensitive and can be induced with thymosin fraction 5 or alpha 1 thymic peptides to express the theta antigen. Enrichment for these suppressor pre-T lymphocytes can be achieved by a one-step density centrifugation, macrophage depletion, or a combination of both procedures. NZB mice, which spontaneously develop an autoimmune disorder, have a suppressor abnormality revealed by this assay system. Upon analysis, they have normal BM pre-T suppressor cells but their spleen cells are refractory to the BM suppressor signal. NZB BM suppressor cells inhibit the response by DBA/2 spleen cells, but DBA/2 BM suppressor cells do not inhibit NZB spleen. This resistance to suppression is a property of the B cell fraction recovered from NZB spleen.     

Abelson virus-infected cells can exhibit restricted in vitro growth and low oncogenic potential.

We have designed a method for growing bone marrow cells infected with Abelson murine leukemia virus which permits examination of target cell growth early after infection. This culture system increases the efficiency of target cell growth by favoring rapid growth of a mixed population of adherent cells in the primary culture. The nonadherent Abelson virus-infected cell populations expressed pre-B-cell differentiation markers characteristic of Abelson virus-transformed cells (mu-heavy chains of immunoglobulin M and terminal deoxynucleotidyltransferase). Early after infection, these cell populations exhibited restricted in vitro and in vivo growth properties which differed from those of an established Abelson virus-transformed cell line, 2M3. These included a marked dependency upon the adherent cell layer for growth and viability, a lower efficiency of agar colony formation, and a lower capacity for tumor production in syngeneic animals. Growth of the early populations could be maintained in the absence of the adherent cell layer by using conditioned medium from long-term adherent cell cultures established in the absence of viral infection. After passage of the populations for several weeks, the in vitro growth properties gradually shifted toward that of the 2M3 cell line. Twelve-week-old populations grew independently of the adherent cell layer and showed an increased efficiency of agar colony formation. These data indicate that many lymphoid target cells exhibit an intermediate transformed phenotype when infected with Abelson virus. Growth of these cells in culture is mediated via a synergistic interaction between intracellular expression of the viral transforming gene and an exogenous growth-promoting activity which can be provided by cultures of adherent bone marrow cells.     

Interleukin 3 is a major negative regulator of the generation of natural killer cells from bone marrow precursors

We have established a bone marrow culture system in which mature natural killer (NK) cells can be generated from inactive precursors by interleukin 2. Recombinant interleukin 3 (IL 3) almost completely blocked the induction of NK cells in this culture system as judged by cytotoxic activity, as well as appearance of cells with NK phenotype. The dose-response curve for inhibition of the generation of NK activity with IL 3 parallelled the growth promoting activity on the strictly IL 3-dependent cell line L/B. The effect of IL 3 was selective for the precursor stage of the NK cell, because mature NK cells were not affected by culture with IL 3 for the same period of time. Moreover, the effect of IL 3 was confined to the first 24 hr of culture, indicating an effect on an early stage of NK cell differentiation. IL 3 did not increase the small normally occurring NK-sensitive population in bone marrow, and did not affect the activity of a variant cytotoxic cell with specificity for adherent target cells, the natural cytotoxic cell. Concomitantly with downregulation of NK cell generation, IL 3 induced strong proliferation in the bone marrow cultures and an increase in the percentage of cells expressing the T cell marker Thy-1. A model for regulation of NK cells based on competition of growth factors for target cells with a common progenitor is discussed.