Stromal cells derived from spleen or bone marrow support the proliferation of rat natural killer cells in long-term culture.

Rat nylon wool nonadherent bone marrow cells were propagated for up to 75 days in co-culture with stromal cells derived from either spleen or bone marrow. Interleukin (IL) 1 enhanced the ability of spleen stroma to support the long-term culture of natural killer (NK) cells, ostensibly by inducing these support cells to synthesize other cytokines. Flow cytometry studies indicated that the nylon wool separation procedure enriched the concentrations of mature NK cells from 7.9% to 38.1% for splenocytes and from 3.8% to 19.5% for bone marrow cells. Analyses of the adherent zones of suspended nylon screen NK cell cultures revealed substantial numbers of large granular lymphocytes that expressed NK 323+/MOM/3F12/F2- phenotypes. The presence of both mature and immature cells of the NK lineage in this matrix was inferred by the presence of both IL-2 receptor (IL-2R) positive and IL-2R negative, and OX-8+ and OX-8- NK 323+ cells over the greater than 4-month experimental period. Suspended nylon screen cultures displayed a greater potential for producing cytolytic cells than either co-cultures of bone marrow nonadherent cells on stroma monolayers or suspension cultures. The large granular lymphocytes produced in suspended nylon screen cultures could be transformed into active killers of YAC-1 targets by IL-2. In contrast to bone marrow nonadherent cells, more splenic nylon-wool-passed cells displayed a mature NK phenotype, but their proliferative potential and ability to be transformed into cytolytic cells by IL-2 decreased rapidly in culture. In the suspended nylon screen culture system, NK cells migrate from the underlying stroma in stages as they mature, retain their cytolytic potential, and manifest a capacity for self-renewal. Cultured cells were routinely dissociated into single cell suspensions via enzyme treatment and were reinoculated onto "fresh" nylon screen/stromal cell templates after passage through nylon wool columns. These co-cultures continued to generate cytolytic cells in numbers greater than those of the initial inoculum.     

Lymphohemopoiesis in culture is prevented by interaction with adherent bone marrow cells from mutant viable motheaten mice

Mice with the recessive "motheaten" (me) or "viable motheaten" (mev) mutations have severe immunologic disturbances and die at an early age. The function of hemopoietic progenitor cells and microenvironmental elements that regulate their growth and differentiation were studied in mev mice with two types of long term bone marrow cultures. Cells from bone marrow of homozygous defective mev/mev mice were non-productive under conditions that normally support replication of stem cells and production of neutrophil granulocytes. Similarly, in a different culture system, lymphocytes were produced from normal littermate, but not mev/mev bone marrow. Initial overgrowth of cells having macrophage-like characteristics occurred in both culture systems with marrow from defective mice. Co-cultures of normal and defective bone marrow cells were always non-productive. In contrast, supernatants of mev/mev bone marrow cultures did not have a detrimental effect on cultures of normal cells, implying that the suppression was cell-associated. Furthermore, there was no evidence for abnormal release of granulocyte or macrophage growth factors in mev bone marrow cultures. A small population of cells in mev/mev bone marrow cultures were morphologically similar to "stromal" cells that support lymphohemopoiesis. Certain culture strategies could be used to enrich for these. mev/mev stromal cells had affinity for normal lymphocytes; however, they did not support lymphocyte growth. The long term bone marrow cultures thus reveal an apparent imbalance in the regulatory mechanisms affected by these single gene mutations. This is manifested by preferential or aberrant growth of one type of adherent cell and a possible functional abnormality of stromal cells. mev mice could provide an ideal model for investigating cell-associated molecules that normally limit progenitor cell replication.     

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.     

Adherent cells (macrophages?) in tumor-bearing mice suppress MLC responses

The spleens of mice bearing transplanted methylcholanthrene (MCA)-induced fibrosarcomas (MCA-1425 and MCA-1460) were shown to contain cells capable of suppressing the generation of cytolytic T lymphocytes (CTL) in mixed leukocyte cultures (MLC). The suppressive activity was first detected 21 days after tumor transplantation. No suppression was seen with lymph node cells taken at the same time as the spleen cells. The cells responsible for the suppressive activity were adherent to nylon wool and plastic dishes and they were not lysed by anti-T-cell serum plus complement. The suppressor cells were phagocytic and were resistant to irradiation (3000 rads) in vitro. Spleen cells from tumor-bearing nude mice were as suppressive as were spleen cells from tumor-bearing conventional mice. We conclude from these findings that T cells were not involved either as inducers or as effectors of the suppression observed, although the responsible adherent cells may have exerted their effect by interacting with a T-suppressor cell population in the MLC mixtures. While spleen cells of tumor-bearing mice were suppressive when added at any time during the first 4 days of a 5-day MLC, they showed no effect on the cytotoxicity of fully differentiated CTL. Indomethacin reversed suppression, suggesting that prostaglandins may have been involved.     

Generation and characterization of purified adherent lymphokine-activated killer cells in mice

During the incubation of murine spleen, lymph node, or bone marrow cells with IL-2 (1000 U/ml) a small percentage of cells became adherent to the surface of plastic tissue culture flasks. After removal of the non-adherent lymphoid cells, plastic adherent lymphokine-activated killer (LAK) cells could be efficiently expanded in the presence of IL-2. Plastic adherent-derived A-LAK cells were characterized by high rates of proliferation and their cytotoxic activity was more than 10 fold higher than LAK cells generated in the bulk (unfractionated) spleen cell cultures. A-LAK cells could be continuously generated from the non-adherent cell population. Using multiple transfers (every 1 to 2 days) of non-adherent LAK cells into new flasks, new rounds of plastic adherent cells were generated with high expansion capability and high levels of cytotoxic activity. Morphologically, A-LAK cells were large granular lymphocyte and phenotypically expressed markers characteristic of NK cells (asialo GM1+, NK1.1+, Qa5+, Ly-6.2+, Thy-1.2+, but negative for Lyt-2.2 and L3T4). A-LAK cells generated from mice of different strains expressing low and high levels of NK cell activity were equally highly cytotoxic. However, A-LAK cells obtained from nude or beige mice had relatively lower levels of cytotoxicity. Stimulation of NK cell activity by poly I:C or inhibition by in vivo or in vitro treatment with anti-asialo GM1 serum did not affect the generation of A-LAK cells. A-LAK cells derived from spleen or bone marrow of C57BL/6 or nude mice treated with anti-asialo GM1 serum were found to be asialo GM1+ suggesting that A-LAK cell could be generated from the asialo GM1- precursor cells. Expansion of plastic adherent A-LAK cells in the presence of IL-2 could provide large numbers of highly purified cytotoxic A-LAK cells suitable for cancer immunotherapy.     

A comparative analysis of the cell surface properties of activated vs endogenous mouse natural killer cells

Mouse natural killer (NK) cells can be activated by interferon and by interferon inducers, such as viruses. In the present report we compared the cell surface properties of the nonactivated "endogenous" NK cells from normal mice with NK cells activated in vivo by acute infection with lymphocytic choriomeningitis virus (LCMV) or in vitro by interferon. Several striking differences were found: 1) in vivo LCMV-activated as well as in vitro interferon-activated NK cells were more adherent to nylon wool columns than the endogenous NK cells; 2) activated NK cells were more EA-monolayer adherent than endogenous NK cells; this adherence could be blocked by streptococcal A protein, indicating that activated NK cells expressed greater Fc receptor mediated adherence; 3) LCMV-activated NK cells that passed through nylon wool columns expressed relatively low EA adherence properties; 4) the sensitivity of NK cells to anti-theta serum + complement treatment was increased in spleen cells late after LCMV infection (day 6 to 7) but not in spleen cells early after infection (day 2 to 3); 5) as shown by centrifugal elutriation, LCMV-activated spleens contained a population of large NK cells that were not present among endogenous spleen cells. In contrast to previous reports, these findings show that there are several distinct changes as a result of both long-term in vivo activation or of short-term in vitro activation of NK cells. These changes might functionally be involved in the increased lytic ability of activated NK cells.     

Non-specific immunosuppression by Cryptococcus neoformans infection

Cryptococcus neoformans-infected animals were found to be immunosuppressed when tested by a variety of assays for immune competence. Primary humoral immune responses and delayed-type hypersensitivity reactions to sheep erythrocytes were suppressed in animals which had been infected for two weeks. Lymphocyte proliferation (LP) assays to sRBC stroma were also significantly diminished at two weeks of infection. Spleen cells of infected mice suppressed the LP response of sRBC immunized, normal mice in vitro. At least a part of the suppression could be attributed to a nylon wool non-adherent cell. Suppressor cells continued to be present in spleen cell suspensions following treatment with anti-T cell serum or anti-immunoglobulin and complement. When infected spleen cells were separated by adherence to plastic, both the adherent and non-adherent fractions exhibited suppressive activity. Incubation of infected spleen cells in tissue culture for 48 hr resulted in the elaboration of soluble immunosuppreessive factors into the tissue culture medium. These data indicated that immune suppression in cryptococcosis can occur as a result of infection with Cryptococcus neoformans, and that at least one mechanism involved is the induction of adherent and non-adherent suppressor cells in the spleens of infected mice.     

Lack of spontaneous and inducible natural killer cell activity in human bone marrow: presence of adherent suppressor cells

Human bone marrow cells collected from ribs of patients undergoing thoracotomy had low or no natural killer (NK) cell activity against K562 in a 4-hour chromium release assay. In vitro overnight treatment with interferon or interleukin 2 of bone marrow cells resulted in no induction or augmentation of NK cell activity. In the presence of adherent bone marrow cells interferon was unable to enhance NK cell activity of blood lymphocytes, although the baseline level of NK cell activity was not suppressed. These results suggest that adherent bone marrow cells regulate the development of active NK cells and that bone marrow components do not provide a favorable environment for the functional differentiation of NK cells.     

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.     

Mechanism of loss of natural killer activity in P815 ascites tumor bearing DBA/2 mice

P815 tumor cells (10(7] were administered intraperitoneally to DBA/2 mice. As the ascites tumor grew in the syngeneic host, a decline leading to a total loss of host spleen natural killer (NK) activity could be demonstrated. Removal of T and B cells or macrophages from the tumor-bearing (TB) mouse spleen cells did not raise the level of NK activity. Spleen cells from TB mice did not inhibit the NK activity of normal spleen cells. Comparable target (YAC cells) binding capacity could be demonstrated in spleen cells derived from normal or TB mice, but interferon failed to significantly stimulate the NK activity of TB mouse spleen cells. In adoptive transfer experiments, transfer of spleen or bone marrow cells from TB mice resulted in the development of significant levels of spleen NK activity in lethally X-irradiated recipient DBA/2 mice. These results indicate that the impairment of NK cell differentiation pathway rather than active suppression at the level of effector cells may be the mechanism of loss of NK activity in P815 TB DBA/2 mice.     

Functional characteristics of a Ficoll-separated mouse bone marrow cell population involved in skin allograft prolongation

Treatment of recipient mice with donor bone marrow cells and anti-lymphocyte serum (ALS) results in extensive skin graft prolongation beyond that achieved in animals given only ALS. In this study, B6AF1 recipient mice were grafted with C3H/He skin on day 0, treated with ALS on days -1 and +2 and infused on day +7 with donor strain (C3H/He) bone marrow cells. Extensive graft prolongation was achieved either with 25 X 10(6) whole bone marrow cells or with 1 X 10(6) lymphoid-like cells derived from donor marrow that sediment at a rate of 3 mm/hr in a 2 to 4% Ficoll gradient at unit gravity. These allograft-prolonging lymphoid-like cells appear not to be CFUs cells, have suppressive activity in in vitro MLC assays, and contain both nylon wool adherent and non-adherent cells. These studies thus show that allograft promoting cells can be isolated from bone marrow utilizing Ficoll gradients. Functional studies suggest that 3 mm/hr sedimenting donor bone marrow suppressor cells may be involved in the induction of allograft prolongation.     

Origin and differentiation of natural killer cells. I. Characteristics of a transplantable NK cell precursor

To study the origin and differentiation of natural killer (NK) cells, we developed an assay for the transplantable precursor of NK(YAC-1) cells present in the bone marrow. Mice were depleted of endogenous NK(YAC-1) cells by injection of anti-asialo GM1 antibody, followed by lethal whole body irradiation. Normal syngeneic bone marrow cells were transplanted into such pretreated mice. Regeneration of NK(YAC-1) activity in the recipient mice was monitored by two different assays: the ability of spleen cells to lyse YAC-1 cells in vitro and the ability to clear i.v. injected, 125IUdR-labeled YAC-1 cells from the lungs. With both assays, a dose-response relationship between the number of bone marrow cells injected and the degree of NK(YAC-1) activity generated could be demonstrated. However, the lung clearance assay appeared superior because the NK regeneration could be detected earlier and with lower numbers of injected marrow cells. With this assay, several characteristics of the NK precursors and their differentiation could be defined. 1) The generation of mature, lytic NK cells from their transplantable precursor requires an intact "marrow microenvironment" in the recipient mice, because differentiation failed to occur in mice rendered osteopetrotic by estradiol treatment. 2) The NK(YAC-1) precursors lack the surface antigens (NK-2.1, asialo GM1, Qa-5, Thy-1) that are characteristically seen on mature NK cells. 3) The NK-precursors could be eliminated from the bone marrow with anti-Qa-2 or anti-H-2 antisera + complement, indicating that these two antigens are expressed on the precursors. The relationship between NK(YAC-1) precursors and multipotent myeloid stem cells (CFU-S) was investigated by utilizing W/Wv and Sl/Sld mutant mice. Bone marrow cells of W/Wv anemic mice, although markedly deficient in CFU-S, have a normal frequency of NK(YAC-1) precursors. Sl/Sld mice that lack a suitable microenvironment for the development of CFU-S allowed normal differentiation of NK(YAC-1) precursors when transplanted with normal bone marrow cells. Together, these data suggest that multipotent myeloid progenitor cells, as defined by the CFU-S assay, and the NK(YAC-1) precursors are not closely related.     

Defective lymphopoiesis in the bone marrow of motheaten (me/me) and viable motheaten (mev/mev) mutant mice. II. Description of a microenvironmental defect for the generation of terminal deoxynucleotidyltransferase-positive bone marrow cells in vitro

We have presented evidence in a previous paper that the development of prothymocytes, pre-B cells, and TdT+ lymphoid precursor cells in the bone marrow of motheaten (me/me) and viable motheaten (mev/mev) mice is defective. In the present study, we have used a selective culture system that supports the generation of rat- and mouse-origin TdT+ bone marrow lymphoid cells in vitro to further investigate the early stages of lymphopoiesis in me/me and mev/mev mice. The results demonstrate that bone marrow stromal cell feeder layers derived from me/me and mev/mev mice do not support the growth of rat TdT+ cells in vitro, whereas stromal cell feeder layers from heterozygous (+/-) littermates and wild type (+/+) control mice do. Moreover, composite feeder layers formed by mixing as few as one part me/me and mev/mev bone marrow cells with 7 to 9 parts +/- littermate bone marrow cells also fail to effectively support the generation of TdT+ cells in vitro. In contrast to me/me and mev/mev mice, other mutant mouse models of autoimmune (NZB, NZB/W), immunodeficient (nu/nu), and hemopoietic (W/Wv, Sl/Sld) disorders form feeder layers that support normal to elevated levels of TdT+ cell growth in vitro. Thus, to date, only the me/me and mev/mev mutant mice have been found to lack the appropriate microenvironment for the generation of TdT+ bone marrow cells. Histologic analysis of the stromal cell feeder layers that are formed in our culture system shows that multilayered cellular patches, which normally are the most active sites of TdT+ cell development in vitro, are absent in feeder layers of me/me and mev/mev cells. Moreover, feeder layers from mev/mev mice contain a population of MAC 1+, basophilic, nonvacuolated, macrophage-like cells; whereas feeder layers from control mice contain MAC 1+, eosinophilic, vacuolated macrophage-like cells. Stromal cell feeder layers formed by mixtures of me/me or mev/mev and control mouse bone marrow cells contain numerous multilayered cellular patches and vacuolated mononuclear cells, but also contain large numbers of basophilic mononuclear cells. These composite feeder layers have a disproportionately reduced capacity to support the generation of TdT+ cells in vitro. Although the stromal microenvironment of me/me and mev/mev bone marrow does not support the growth of TdT+ cells in vivo or in vitro, the bone marrow from these mutant mice contains detectable numbers of pre-TdT+ cells. Thus, when cultured on normal mouse feeder layers, mutant mouse bone marrow rapidly generates TdT+ cells in vitro, albeit at significantly reduced levels as compared to +/- littermate controls.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cellular mechanisms of suppression of T-lymphocyte proliferation by lung cells in experimental tuberculosis]

The ability of interstitial lung cells from mice, infected with Mycobacterium tuberculosis H37Rv, to suppress proliferative responses of immune lymphocytes to mycobacterial (PPD) and unrelated (Staphylococcus aureus cytoplasm) antigens was studied. Two types of suppression were observed: the specific one, which was characteristic of the PPD-response only; and non-specific. The latter was mediated mainly by prostaglandins, since it could be abolished by indomethacin. Both types of suppression depended on the presence of plastic and nylon wool adherent phagocytes from infected lung. Though the depletion of T or B lymphocytes from the lung cell population have not abrogated the suppressive effect, some intercellular interactions were required for antigen-specific suppression, since the presence of nylon wool adherent cells in the population of responder lymph node cells was necessary for its development.     

Adriamycin-induced activation of NK activity may initially involve LAF production

C3HeB/FeJ spleen cells (unseparated or passaged over nylon wool columns) were cultured overnight (1-2 X 10(6) cells/microwell) in the presence and absence of resident or ADM-induced PEC and anti-YAC-1 (4h) NK activity was determined. The addition of resident PEC to spleen cells had little effect on NK activity. However, the addition of ADM-elicited PEC (10 mg/kg, IP, day -1 and day -5) to spleen cells prior to culture significantly augmented NK activity. If ADM-induced PEC were treated with carbonyl iron prior to coculture with spleen cells, augmentation of anti-YAC-1 activity was not observed. This suggested that ADM-activated macrophages augmented cultured splenic NK activity. Supernatants from overnight-cultured resident or ADM-induced adherent PEC were then prepared, dialyzed (to remove ADM), and tested for mitogenic activity or cocultured with spleen cells overnight. ADM-induced adherent PEC supernatants stimulated the proliferation of murine thymocytes (both LAF and IL-2 also stimulate) but not cultured CTL (only IL-2 stimulates). ADM-induced adherent PEC supernatants (as well as LAF, IL-2, and IFN) augmented overnight-cultured C3HeB/FeJ splenic NK activity. However, only IL-2 and IFN could augment overnight-cultured athymic BALB/c . nu/nu splenic NK activity. This suggested that ADM-elicited macrophages produce LAF which may act directly on NK cells or, more likely, may induce T cells to produce IL-2, IFN, or both.     

Physiology of natural killer cells. In vivo regulation of progenitors by interleukin 3

Adoptive transfer of bone marrow cells to syngeneic lethally irradiated C57BL/6 mice was used to study the maturation of natural killer (NK) cells from their progenitors. The NK progenitor cell was found to be asialomonoganglioside-negative, (aGM1-) Thy-1-, NK-1-, Ly-1-, Ly-2-, and L3T4-. The NK cells emerging from the bone marrow grafts were aGM1+, NK-1+, Thy-1+/-, Ly-1-, Ly-2-, and L3T4- and to have a target specter similar to that of NK cells isolated from the spleen of normal mice. The regulatory role of interleukin 2 (IL-2) and interleukin 3 (IL-3) for the maturation of NK cells was examined by exposure of the bone marrow cells to the lymphokines in vitro before bone marrow grafting or by treatment of bone marrow-grafted mice with lymphokines through s.c. implanted miniosmotic pumps. IL-3 antagonized the IL-2-induced maturation of NK cells in vitro and strongly inhibited the generation of NK cells after adoptive transfer of bone marrow cells in vivo. The suppressive effect of IL-3 was evident throughout the treatment period (8 or 16 days) but was apparently reversible because NK activity returned to control levels within 8 days after cessation of treatment. The inhibition of cytotoxic activity was accompanied by a reduced appearance of cells with the NK phenotypic markers aGM1 or NK-1, indicating that not only the cytotoxic activity of NK cells but also their actual formation was inhibited. Concomitantly, a moderate increase in cells expressing the T cell marker L3T4 and an increased proliferative response to the T cell mitogen concanavalin A was observed. A direct estimate of the effect of IL-3 on the frequency of NK cell progenitors was obtained by limiting dilution analysis of bone marrow cells at day 8 after bone marrow transplantation. The estimated minimal frequency of NK cell progenitors was reduced from 1/11,800 in control to 1/41,900 in IL-3-exposed mice. IL-3 may take part in the homeostasis of NK cells by the down-regulation of their progenitors.     

A stromal cell line from myeloid long-term bone marrow cultures can support myelopoiesis and B lymphopoiesis

The production of B lymphocytes and myeloid cells occurs in the bone marrow in association with a supporting population of stromal cells. To determine whether these processes are dependent upon the same or different populations of stromal cells, stromal cell lines were generated from the adherent layer of a Dexter type long-term bone marrow culture. These cultures support myeloid cells and their precursors, a B cell precursor, and the adherent layer cells with support B cell differentiation under appropriate conditions. Two of the lines examined, S10 and S17, express class I histocompatibility antigens but not other hemopoietic cell surface determinants such as Thy-1, Lyt-1, Ig, Ia, Mac-1, or BP-1. Both lines could support myelopoiesis under Dexter conditions upon seeding with nylon wool-passed bone marrow. The nylon wool passage depletes stromal cells capable of forming adherent layers in vitro but retains hemopoietic precursors. The number of cells and colony-forming units-granulocytes/macrophages in the nonadherent cell population recovered 3 wk post-seeding had increased 19-fold and 10-fold, respectively, in the reseeded cultures of S10 and S17. After 3 wk of growth in Dexter conditions, the reseeded cultures were transferred to conditions optimal for B cell differentiation described by Whitlock and Witte. After 4 wk of growth, hemopoietic cells were consistently recovered from S17 cultures but not those of S10. A proportion of these cells from S17 cultures expressed the 14.8 antigen and were surface IgM positive. Surviving hemopoietic cells present in cultures of S10 were primarily macrophages. These findings indicate that S17 but not S10 can support both myelopoiesis and B lymphopoiesis and suggest that one stromal cell population has the capacity to form a hemopoietic microenvironment for both lineages.     

Specific, transient suppression of the immune response by HGG tolerant spleen cells. II. Effector cells and target cells.

In a previous report, it was shown that spleen cells from mice made tolerant to human gamma-globulin (HGG)5 could specifically inhibit the immune response of normal spleen cells after adoptive transfer to lethally irradiated recipients. However, that report also showed that the suppressive activity was only transiently associated with tolerant spleen cell populations. It was concluded from those experiments that while suppressive activity could be demonstrated in tolerant spleen cells under certain conditions, such activity was not obligatory for the maintainance of the tolerant state. The experiments presented here were performed to determine the nature of the effector cell(s) and the target cell(s) involved in this system of suppression of the immune response. Treatment of cells from tolerant animals with anti-thymocyte serum and complement to remove thymus-derived (T) cells completely abrogated suppresive activity. Removal of adherent cells from tolerant spleen cells by passage over glass wool columns resulted in partial loss of the suppression. The inhibitory activity of the suppressor cells was resistant to 900 R irradiation regardless of whether the tolerant spleen cells were irradiated before or after adoptive transfer. The cellular target(s) for the supprssor cells was examined by using lipopolysaccharide (LPS) as an alternative source of helper activity for the response to HGG. LPS, injected at the time of the initial antigenic challenge of mice that had been reconstituted with tolerant and normal spleen cells, prevented the expression of suppression against bone marrow-derived (B) cells. However, when LPS was presented only at the time of secondary antigenic challenge, it was unable to overcome suppression of the immune response of reconstituted recipients. Thus, LPS could produce a state where the B cells were resistant to suppression, but LPS could not rescue the responsiveness of B cells once the cells in the reconstituted recipient had been suppressed. In addition, the immune response to both the hapten dinitrophenol (DNP) and the carrier (HGG) were suppressed when recipients of tolerant and normal spleen cells were challenged with DNP6HGG. This indicates that T helper cells are also a target for suppression. The results presented in this paper are discussed in relation to a possible mechanism of suppression which proposes that suppressive activity represents the induction of tolerance in immunologically competent cells by HCG which is closely associated with the tolerant spleen cells.     

Origin and differentiation of natural killer cells. II. Functional and morphologic studies of purified NK-1.1+ cells

Cells bearing the NK-specific marker NK-1.1 were purified from mouse spleens by utilizing a monoclonal anti-NK-1.1 antibody and cell sorting. In normal adult mice, all of the splenic NK activity against YAC-1 cells was found in the NK-1.1+ fraction, whereas NK-1.1- cells were depleted of NK activity. The NK activity of sorted NK-1.1+ cells was enriched 15- to 30-fold over unfractionated spleen cells. Light and electron microscopic studies of purified NK-1.1+ cells showed a homogeneous population of cells, each containing one to four cytoplasmic granules. Mice whose bone marrow has been destroyed by chronic exposure to 17-beta-estradiol have very low NK activity. However, spleen cells of estradiol-treated mice contained a normal frequency of NK-1.1+ cells which bound to YAC-1 cells, but failed to lyse them even after purification and subsequent exposure to interferon-alpha/beta in vitro. It appears, therefore, that in the absence of intact bone marrow, NK-1.1+ cells may be arrested in a nonlytic and interferon-unresponsive state. Spleens of neonatal mice which have low NK activity were analyzed to ascertain whether immature NK-1.1+ cells, similar to those found in estradiol-treated mice, could be demonstrated. Spleens of 8- to 9-day-old mice also contained NK-1.1+ cells which had very low NK activity even after purification. Sorted NK-1.1+ cells were examined for cytotoxicity in mice whose NK activity was suppressed by pretreatment with Corynebacterium parvum (-15 days). In contrast to cells from estradiol-treated and neonatal mice, NK-1.1+ from mice treated with C. parvum had normal functional activity. Similarly, although NK activity of unfractionated bone marrow cells is low, sorted NK-1.1+ cells were greatly enriched for lytic activity. Thus, we conclude that cell sorting with monoclonal anti-NK-1.1 antibody provides a powerful tool for examining the mechanisms underlying various states of low NK activity, and there exist NK-1.1+, nonlytic, interferon-unresponsive cells which apparently require an intact marrow microenvironment for differentiation into mature, lytic NK cells.     

Immunoregulation in disseminated histoplasmosis: Characterization of splenic suppressor cell populations

Potent immunosuppressor cell activity was induced during the course of disseminated histoplasmosis in C3H/Anf mice. Spleen cells from infected mice severely suppressed the primary antibody response in vitro of normal syngeneic spleen cells to both a T-dependent antigen (sheep red blood cells) and a T-independent antigen (trinitrophenyl-lipopolysaccharide) at Weeks 1 and 3 of infection, respectively. Likewise, marked suppressor cell activity was present within lymph nodes. In a kinetic study, suppressor activity was detected first on Day 2 and increased to the maximum level on Day 4 after inoculation of Histoplasma capsulatum. Two populations of spleen cells express suppressor function in this model. One population, identified as T cells, was nonadherent to nylon wool columns; its suppressor capacity was abolished by anti-Thy 1 and reduced greatly by low-dosage X-irradiation (500 R). Cells of the second suppressor population had macrophage-like properties; although poorly adherent to plastic surfaces, they adhered to nylon wool columns and could be removed from spleen cell suspensions by carbonyl iron treatment; high-dosage X-irradiation (3000 R) and mitomycin C treatment failed to abrogate suppression by these cells.