Enrichment of the murine natural killer (NK) and mitogen induced cellular cytotoxicity (MICC) cells using preparative free-flow high voltage electrophoresis.

A procedure using preparative free-flow high voltage electrophoresis is described for the fractionation of murine spleen and bone marrow cells so as to obtain cell subpopulations that are either enriched in or depleted of "natural killer" (NK) cells and "mitogen-induced cellular cytotoxicity" (MICC) effector cells. A nearly three fold enrichment in the NK and MICC activities of spleen cells was achieved. The enrichment in these cells could be further increased if the phagocytic cells were removed prior to electrophoresis. When bone marrow cells were fractionated a two and a half fold increase of NK activity, and a one and a half fold enrichment of MICC activity was achieved. In both cases, other fractions were nearly devoid of NK and MICC activity. The cell recovery after electrophoresis averages 70% of the cells applied, and at least 90% of these cells were viable. MICC and NK effector cells could not be separated to a useful extent electrophoretically but were found to be separable using Sephadex C-10 gel filtration columns. The MICC but not the NK cells were retained on these columns.     

Mitogen induced cellular cytotoxic activity as a monocyte function in human peripheral blood preparations

The effector cell population in man responsible for mitogen induced cellular cytotoxicity (MICC) of chicken erythrocytes was investigated using several separation techniques, including free flow electrophoresis. Electrophoresis produced substantial monocyte enrichment in some fractions with substantial depletion in others. MICC activity was seen to correlate with monocyte content in these fractions. Furthermore, removal of phagocytic cells with carbonyl iron and removal of adherent cells on plastic petri dishes depleted preparations of MICC activity. Thus it is suggested that under conditions described in this paper, the effector cell of the MICC assay in man appears to be a monocyte. This MICC effector cell was shown to be different from the effector cell in the natural killing (NK cells) of RPMI 6410 cells.     

Natural killer cell activity in mouse aggregation chimeras

The activity of natural killer (NK) cells in spleen against syngeneic and allogeneic tumor cells was studied by the use of tetraparental mouse chimeras. Chimeras were produced by aggregation of early embryos of histoincompatible mouse strains of “high” and “low” NK cell activity. NK activities of spleen cells were assayed in vitro by the ⁵¹Cr-release method. Coat color distribution and isozymal analysis (glucose-phosphate isomerase) of several lymphoid organs (thymus, lymph nodes, and bone marrow) revealed a predominant share of the “high”-NK-reactive genotype in the chimeras. However, the cellular NK activity against two target cell lines differing in their susceptibility to lysis was significantly lower in chimeras than in the “high”-reactive strain. Addition of “low”-NK spleen cells or of NH₄Cl-inactivated “high”-NK spleen cells to “high”-NK spleen cells inhibited their cytolytic activity. Possible mechanisms of the suppression of the cytolytic capacity of NK cells in chimeras are discussed.     

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.     

The nature of the effector cells mediating mitogen-induced cellular cytotoxicity (MICC) and antibody-dependent cellular cytotoxicity (ADCC).

The nature of the cell types capable of mediating mitogen-induced cellular cytotoxicity (MICC) and antibody-dependent cellular cytotoxicity (ADCC) was investigated utilizing effector cells from athymic nude and euthymic heterozygous control littermate mice as well as Sephadex anti-Fab immunoabsorbent column purified spleen cell populations from normal (CS7BL/6) mice. Chicken erythrocytes (CRBC) and the mouse lymphoma, EL-4, were used as target cells in both cytotoxicity assays. MICC utilizing CRBC targets was mediated by several effector cell types whereas MICC utilizing EL-4 lymphoma targets was T-cell dependent. ADCC against both CRBC and EL-4 lymphoma targets occurred independently of the presence of T-cells. In addition, effector cell populations incapable of mediating MICC against EL-4 lymphoma targets were capable of mediating ADCC against the same EL-4 targets. Thus, utilizing the appropriate target cells, EL-4 but not CRBC, a sharp distinction can be made between the effectors for ADCC and MICC: ADCC is T-cell independent while MICC is dependent on the presence of mature thymus-derived cells. Furthermore these studies demonstrate that the nature of the target cell employed in MICC and ADCC reactions plays a critical role in defining the types of effector cells capable of mediating these cytotoxicity reactions.     

Simultaneous development of antibody-dependent cellular cytotoxicity (ADCC) and natural killer (NK) activity in irradiated mice reconstituted with bone marrow cells

Spleen cells from irradiated, bone marrow-reconstituted mice were tested for their ability to mediate antibody-dependent cellular cytotoxicity against P815 target (ADCC-P815), ADCC against sheep red blood cells (ADCC-SRBC), and natural killer (NK) activity judged as YAC-1 lysis at different times after bone marrow reconstitution. Donor-derived ADCC-P815 effectors were found to appear in the spleens 10-12 days after bone marrow reconstitution simultaneously with the appearance of donor-derived NK cells. NK cells recently derived from bone marrow are known to express the Thy-1 antigen; the phenotype of the "early" ADCC-P815 effectors was found to be the same as that of NK cells, i.e., Thy-1+, asialo-GM1+. These data suggest that ADCC-P815 effector cells belong to the NK cell population. ADCC-SRBC, in contrast to ADCC-P815 and NK activity, was already high on Day 7 after bone marrow reconstitution. However, it was mediated partly by recipient-derived effectors. ADCC-SRBC effectors were characterized to be different from ADCC-P815 effectors.     

In vitro modulation of mouse natural killer (NK) cell activity by the serum thymic factor (FTS)

Previous studies indicated that the serum thymic factor (FTS) could modulate in vivo the level of splenic natural killer (NK) cell activity in mice. The present report shows that such an effect is also observed after a short term in vitro incubation of the effector cells with FTS. The regulatory effects of FTS result in an increase or a decrease of the splenic NK cell cytotoxicity depending upon the age and the mouse strain. Furthermore, FTS is able to enhance the NK cell activity of thymus and bone marrow cells which are known to be weakly reactive in NK cytotoxicity. Depletion experiments demonstrated that the FTS-induced increase of NK cell activity was not mediated by Thy 1+ cells nor macrophages, thus suggesting a direct action of FTS on the effector cells. Comparative studies using other thymic hormones revealed similar patterns of reactivity. These results favor the hypothesis of a close relationship between the thymus and NK cells.     

Mitogen induced cellular cytotoxicity (MICC) in multiple myeloma.

Mitogen induced cellular cytotoxicity (MICC) was noted to be markedly increased in patients with multiple myeloma as compared to normal controls and to patients with chronic lymphocytic leukemia (CLL). Enhanced MICC was present at various effector-to-target cell ratios and at several mitogen concentrations. Removal of adherent, phagocytic cells by carbonyl iron, glass wool, or rayon columns abolished the MICC response from the peripheral blood of both multiple myeloma patients and normal controls. Thus, the effector cell mediating MICC may be monocytic in origin and closely resembles the suppressor cell for immunoglobulin synthesis described in patients with multiple myeloma. Our data suggest that the MICC assay with chicken red blood cells as targets may provide a convenient method for identifying pathologic conditions where this cytotoxic effector cell population plays an active role.     

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.     

OK-432 stimulates primary production and activity of murine natural killer cells

Although many immunostimulants have been shown to increase the lytic activity of natural killer (NK) cells in the periphery, little is known about their effects on NK cells in the bone marrow, the primary site of NK production. In the experiments reported here, we tested OK-432, a pharmaceutical preparation of Streptococcus pyogenes, for its effects on both the primary production and lytic activity of NK cells in C57BL/6J mice. NK activity in bone marrow cells (BMC) and spleen cells (SC) was significantly increased following intravenous administration of OK-432, peaking on day 2 in BMC and on day 3 in SC. Concomitantly, there were marked changes in the cellularity in the two compartments. Bone marrow cellularity fell significantly on day 1 post-OK-432 and then gradually returned to normal, whereas spleen cellularity rose rapidly and remained elevated. As a consequence, the total NK activity (per femur or per spleen) was significantly increased at 48-96 h after administration of OK-432. The target specificity was unchanged. The phenotype of NK cells in BMC as determined by cytotoxic depletion was unchanged by OK-432, but splenic NK activity shifted to a 'less mature' phenotype, intermediate between that of normal BMC and SC. Cytokinetic studies using 3H-TdR revealed an increase in the production of NK cells in the bone marrow following administration of OK-432. Proliferating NK cells also appeared in the spleen. Whether these were recently produced NK cells from the bone marrow that still retained the ability to proliferate or mature NK cells that were stimulated into cell cycle cannot be determined from these experiments. These data are the first to directly demonstrate the modulation of the primary production of NK cells by an immunologically active drug.     

Mechanisms of lymphocytic choriomeningitis virus-induced hemopoietic dysfunction.

Results of this study showed that lymphocytic choriomeningitis virus infection causes a marked activation of natural killer (NK) cells not only in the spleen but also in the bone marrow. This activity reached its peak at about day 3 of infection and declined after days 6 to 7. Enhanced NK cell activity was found to correlate with decreased receptivity for syngeneic stem cells in bone marrow and spleen, with the notable exception that decreased receptivity persisted longer in bone marrow. Treatment of infected recipients with anti-asialo GM1 (ganglio-N-tetraosylceramide) significantly increased the receptivity for syngeneic hemopoietic cells. These findings are consistent with the hypothesis that NK cell activation causes rejection of syngeneic stem cells, thus resulting in hemopoietic depression. To understand the mechanisms behind the prolonged decrease in bone marrow receptivity (and bone marrow function in the intact mouse) mentioned above, we followed the changes in the number of pluripotential stem cells (CFU-S) circulating in the peripheral blood and in endogenous spleen colonies in irradiated mice, the limbs of which were partially shielded. It was found that following a marked early decline, both parameters increased to normal or supranormal levels at about day 9 after infection. Because the bone marrow pool of CFU-S is only about 20% of normal at this time after infection, a marked tendency for CFU-S at this stage in the infection to migrate from the bone marrow to the spleen is suggested. It seems, therefore, that as NK cell activity declines, the spleen regains the ability to support growth of hemopoietic cells and the bone marrow resumes an elevated export of stem cells to the spleen. This diversion of hemopoiesis could explain both the long-standing deficiencies of the bone marrow compartment and the prolonged decrease in the receptivity of this organ.     

Suppression of natural killer activity in human blood and bone marrow cultures by bone marrow-adherent OKM1-positive cells

Maintenance and regulation of natural killer (NK) cell activity in human bone marrow cultures were studied using K562 leukemia cells as targets. Culture of bone marrow cells in medium supporting long-term generation of myeloid cells resulted in a rapid loss of NK activity in 1-3 days. In contrast, antibody-dependent cytotoxicity to an NK-resistant tumor was maintained for more than 7 weeks. Horse serum, a component of the myelopoietic culture medium, was found to diminish NK cytotoxicity of blood and bone marrow cultures whereas hydrocortisone supplement did not. In addition, an adherent cell is present in bone marrow which greatly inhibits NK activity. Nonadherent bone marrow cells exhibited higher cytotoxicity than unfractionated cells at all days of culture; adherent cells were not cytotoxic to K562. Purified adherent marrow cells inhibited the cytotoxic capacity of nonadherent blood or marrow mononuclear cells during coculture. Indomethacin, an inhibitor of protaglandin synthesis, augmented levels of NK activity in cultures of bone marrow cells, indicating that macrophages may be suppressing this effector function via prostaglandins. Further identification of the adherent suppressor cells came from experiments in which suppression was prevented by treatment of the adherent cells with monoclonal OKM1 antibody plus complement. This study shows that bone marrow-adherent OKM1-positive cells, presumably macrophages, negatively regulate NK activity, and it defines conditions for analysis of the generation and/or positive regulation of NK cells in human bone marrow.     

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.     

Studies on the mechanism of natural killer cytotoxicity. III. Activation of NK cells by interferon augments the lytic activity of released natural killer cytotoxic factors (NKCF)

The mechanism by which interferon (IFN) pretreatment of effector cells augments natural killer (NK) cell-mediated cytotoxicity (CMC) was examined by determining whether IFN has any effect on the production of natural killer cytotoxic factors (NKCF). NKCF are released into the supernatant of co-cultures of murine spleen cells and YAC-1 stimulator cells, and their lytic activity is measured against YAC-1 target cells. It was demonstrated that pretreatment of effector cells with murine fibroblast IFN or polyinosinic-polycytidylic acid (pIC) resulted in the release of NKCF with augmented lytic activity. Evidence indicated that the IFN-induced augmentation of NKCF activity required protein synthesis during the IFN pretreatment period, because concurrent pretreatment with both IFN and cycloheximide abrogated the IFN effect. Protein synthesis, however, is not required for the production of base levels of NKCF because emetine pretreatment of normal spleen cells did not result in a decrease in NKCF production. Furthermore, substantial levels of NKCF activity could be detected in freeze-thaw lysates of freshly isolated spleen cells. Cell populations enriched for NK effector cells, such as nylon wool-nonadherent nude mouse spleen cells, produced lysates with high levels of NKCF activity, whereas lysates of CBA thymocytes were devoid of NKCF activity. Pretreatment of spleen cells with either IFN or pIC resulted in an augmentation of the NKCF activity present in their cell lysates. Taken altogether, these findings suggest that freshly isolated NK cells contain preformed pools of NKCF. Pretreatment of these cells with IFN causes de novo synthesis of additional NKCF and/or activation of preexisting NKCF. According to our model for the mechanism of NK CMC, target cell lysis is ultimately the result of transfer of NKCF from the effector cell to the target cell. The evidence presented here suggests that the IFN-induced augmentation of NK activity could be accounted for by an increase in the synthesis, activation, and/or release of NKCF.     

Evidence for extrathymic development of TNK cells. NK1+ CD3+ cells responsible for acute marrow graft rejection are present in thymus-deficient mice.

The predominant mechanism responsible for acute specific rejection of allogeneic and parental bone marrow by irradiated mice is due to a cell (TNK) that expresses the NK cell surface markers NK1 and ASGM1 as well as TCR. Here we analyze the question as to whether TNK cells require a functional thymus for their development. Using adoptive cell transfer assays, evidence is presented that, as is the case in normal mice, NK1+ CD3+ effector cells are responsible for rejection in thymus-deficient nude mice and that the specificity of rejection is indistinguishable from that of normal mice. To reveal the presence of TNK cells in the spleen of nude mice, double staining for NK1 and CD3 followed by FACS analysis was done. It is shown that NK1+ CD3+ cells are present in the spleens of nude but not euthymic mice, suggesting that the lack of a functional thymus stimulates either Ag expression or the number of TNK cells. In support of this finding, the treatment of irradiated marrow reconstituted mice with cyclosporin A leads to the appearance of TNK cells in the spleen. The relative efficiency of spleen cells from nude and cyclosporin A-treated mice to transfer resistance in adoptive cell transfers was assessed and found to be higher than that of normal spleen, consistent with the higher frequency of these cells in thymus-defective mice. The fate of NK1+ CD3+ cells subsequent to stimulation with an allogeneic marrow graft indicates that these cells proliferate in nude mice without gaining cytolytic activity. In euthymic mice, however, NK1+ CD3+ cells appear transiently but disappear in favor of CD4+ and CD8+ cells that proliferate in response to an allogeneic marrow graft. The CD8+ cells express cytolytic activity with specificity similar to that of the acute rejection mechanism, consistent with the suggestion that TNK cells differentiate into CD8+ killer cells. The reason why TNK cells in nude mice fail to differentiate into CD8+ CTL is explained by the lack of Th cells.     

Characterization of cytotoxic cells generated from in vitro cultures of murine bone marrow cells

Bone marrow cells cultured for 5-6 days generate cytotoxic activity against a number of natural killer (NK)-susceptible tumor cells. In this study, these bone marrow cytotoxic cells were compared to cells with NK activity obtained either from spleen cells activated in vitro with interferon (IFN-alpha/beta) or mitogen or from peritoneal exudate cells (PEC) obtained 4 days after bacillus Calmette-Guerin (BCG) infection. Splenic and PEC cytotoxic cells were shown to be Thy 1.2+, NK 1.1+, Asialo GM+1, Lyt 1.2-, Lyt 2.2-. In contrast, bone marrow cytotoxic cells were Thy 1.2+, NK 1.1-, Lyt 1.2-, Lyt 2.2- and expressed low levels of Asialo GM1 antigen (Asialo GM +/- 1). Precursor cells for bone marrow cytotoxic activity were shown to be Thy 1.2-, NK 1.1-, Lyt 1.2-, Lyt 2.2- but also expressed low levels of Asialo GM1 antigen (Asialo GM +/- 1). Cytotoxic activity for both bone marrow and spleen cells peaked in the low-density fractions of discontinuous Percoll density gradients. The cytotoxic activity of these bone marrow cells was augmented by pretreatment with IFN (-alpha/beta, -gamma) or soluble factors (IFN free) from activated EL-4 thymoma cells. Surprisingly, the ability of bone marrow cells to generate high levels of cytotoxic activity following in vitro culture appeared to be associated primarily with mice which were of the H-2b haplotype.     

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.     

A novel NKR-P1B(bright) NK cell subset expresses an activated CD25(+)CX(3)CR1(+)CD62L(-)CD11b(-)CD27(-) phenotype and is prevalent in blood, liver, and gut-associated lymphoid organs of rats

The inhibitory NKR-P1B receptor identifies a subset of rat splenic NK cells that is low in Ly49 receptors but enriched for CD94/NKG2 receptors. We report in this study a novel NKR-P1B(bright) NK subpopulation that is prevalent in peripheral blood, liver, and gut-associated lymphoid organs and scarce in the spleen, peripheral lymph nodes, bone marrow, and lungs. This NKR-P1B(bright) NK subset displays an activated phenotype, expressing CD25, CD93, CX(3)CR1 and near absence of CD62-L, CD11b, and CD27. Functionally, NKR-P1B(bright) NK cells are highly responsive in terms of IFN-γ production and exert potent cytolytic activity. They show little spontaneous proliferation, are reduced in numbers upon in vivo activation with polyinosinic:polycytidylic acid, and have poor survival in ex vivo cytokine cultures. Our findings suggest that NKR-P1B(bright) NK cells are fully differentiated effector cells that rapidly die upon further activation. The identification of this novel rat NK cell subset may facilitate future translational research of the role of distinct NK cell subsets under normal physiological conditions and during ongoing immune responses.     

Further characterization of PNK-E: a monoclonal antibody enhancing porcine natural killer cell activity

Monoclonal antibody PNK-E binds to approximately 15% of porcine peripheral blood lymphocytes (PBL) which are PT4 negative and PT8 positive. When cells from tissues of adult pigs are treated with PNK-E, enhancement of natural killer (NK) cell activity is observed from PBL and spleen cells, and a dramatic induction of NK activity is observed from bone marrow cells. With cells derived from tissues of neonatal piglets, PNK-E induces NK activity from PBL and bone marrow cells. To investigate the mechanism of PNK-E-mediated enhancement of NK, proliferation assays, calcium-pulse assays, single-cell assays, and kinetic analyses were performed. PNK-E did not induce proliferation of PBL. PNK-E could be added as late as 30 min prior to termination of Ca(2+)-pulse assays and still enhance NK activity. Using kinetic analysis PNK-E was found to increase the rate of NK lysis (Vmax) and rate of lytic programming per NK cell (k2). In addition, results from single-cell assays indicate that PNK-E activates a population of normally inactive effector cells. These results indicate that PNK-E enhances the lytic capacity of mature NK cells and induces a population of nonlytic cells to become highly cytolytic cells. Furthermore, the enhancing effects are immediate and do not require an induction period. Thus, PNK-E recognizes and activates a unique triggering molecule that is present on NK cells.     

Roles for common cytokine receptor gamma-chain-dependent cytokines in the generation, differentiation, and maturation of NK cell precursors and peripheral NK cells in vivo

NK cells differentiate in adult mice from bone marrow hemopoietic progenitors. Cytokines, including those that signal via receptors using the common cytokine receptor gamma-chain (gamma(c)), have been implicated at various stages of NK cell development. We have previously described committed NK cell precursors (NKPs), which have the capacity to generate NK cells, but not B, T, erythroid, or myeloid cells, after in vitro culture or transfer to a fetal thymic microenvironment. NKPs express the CD122 Ag (beta chain of the receptors for IL-2/IL-15), but lack other mature NK markers, including NK1.1, CD49b (DX5), or members of the Ly49 gene family. In this report, we have analyzed the roles for gamma(c)-dependent cytokines in the generation of bone marrow NKP and in their subsequent differentiation to mature NK cells in vivo. Normal numbers of NKPs are found in gamma(c)-deficient mice, suggesting that NK cell commitment is not dependent on IL-2, IL-4, IL-7, IL-9, IL-15, or IL-21. Although IL-2, IL-4, and IL-7 have been reported to influence NK cell differentiation, we find that mice deficient in any or all of these cytokines have normal NK cell numbers, phenotype, and effector functions. In contrast, IL-15 plays a dominant role in early NK cell differentiation by maintaining normal numbers of immature and mature NK cells in the bone marrow and spleen. Surprisingly, the few residual NK cells generated in absence of IL-15 appear relatively mature, expressing a variety of Ly49 receptors and demonstrating lytic and cytokine production capacity.