Cytohesin-associated scaffolding protein (CASP) is involved in migration and IFN-γ secretion in Natural Killer cells

Natural Killer (NK) cells are highly mobile, specialized sub-populations of lymphocytic cells that survey their host to identify and eliminate infected or tumor cells. They are one of the key players in innate immunity and do not need prior activation through antigen recognition to deliver cytotoxic packages and release messenger chemicals to recruit immune cells. Cytohesin associated scaffolding protein (CASP) is a highly expressed lymphocyte adaptor protein that forms complexes with vesicles and sorting proteins including SNX27 and Cytohesin-1. In this study we show that by using stably integrated shRNA, CASP has a direct role in the secretion of IFN-γ, and NK cell motility and ability to kill tumor cells. CASP polarizes to the leading edge of migrating NK cells, and to the immunological synapse when engaged with tumor cells. However, CASP is not associated with cytotoxic granule mediated killing. CASP is a multi-faceted protein, which has a very diverse role in NK cell specific immune functions.     

Natural killer cell number and function in the spontaneously diabetic BB/W rat

The BB/W rat provides a good model of spontaneous autoimmune diabetes. Diabetes-prone (DP) rats have a virtual lack of OX 8+ OX 19+ T cytotoxic/suppressor cells in peripheral blood lymphocytes (PBL) and spleen, suggesting that the OX 8+ OX 9- natural killer (NK) cells are the predominant cytotoxic cell in this animal. In this study, we have shown that rat NK cells belong to the OX 8+ OX 19- asialo GM1 bright population, and that rat NK cell function may be depleted in vivo by administration of OX 8 antibody. Furthermore, evidence is provided to indicate that NK cell number and activity are enhanced on a per cell basis in DP rats as compared to the diabetes-resistant W line rat. DP rats had about threefold more NK cells than did W-line rats. The cytotoxic activity mediated by spleen and PBL against the YAC-1 target generally correlated with the relative number of cells having the OX 8+ OX 19- phenotype. DP lymphocytes mediated low levels of cytolytic activity against the relatively resistant NK target cell K562. To more directly compare the activity of W-line and DP NK cells, spleen NK cells were isolated by flow sorting of the OX 8+ OX 19- population. At a 5:1 E:T ratio, DP OX 8+ OX 19- cells elicited 21% +/- 3 specific lysis and W-line cells elicited 7% +/- 2 specific lysis. To determine whether the elevated levels of NK cells and NK cell activity in DP rats were a consequence of NK cell proliferation, spleen cells were size-separated by centrifugal elutriation. The NK cell activity was predominantly mediated by small to medium-size lymphocytes and not blast-size enriched populations. Moreover, when the DNA content of splenic OX 8+ cells was measured, 98% of the cells were in the G0-G1 phase of the cell cycle. These data indicate that NK cell number and activity are elevated in DP rats, and support a role for NK cells in the pathogenesis of BB/W diabetes.     

Flow cytometric analysis reveals the presence of asialo GM1 on the surface membrane of alloimmune cytotoxic T lymphocytes

Previous studies have demonstrated that natural killer (NK) cells express the glycolipid asialo GM1, as evidenced by the sensitivity of NK cells to treatment with anti-asialo GM1 serum and complement. Because alloimmune cytotoxic T lymphocytes (CTL) were found to be insensitive to treatment with anti-asialo GM1 serum and complement, it was concluded that asialo GM1 is expressed by NK but not by CTL. However, fluorescence studies indicated that a significant proportion of peripheral T cells did express asialo GM1. Flow cytometric studies were undertaken to determine the extent to which alloimmune CTL express asialo GM1. Affinity-purified, monospecific IgG anti-asialo GM1 antibodies were used to label cells from mixed lymphocyte cultures. Separation of asialo GM1-positive and -negative fractions by cell sorting revealed that the majority of CTL activity resides in the asialo GM1-positive population. When these studies are compared with similar studies of splenic NK activity, it is apparent that, despite the relative insensitivity of CTL to treatment with anti-asialo GM1 and complement, both CTL and NK activity are enriched in the asialo GM1-positive cell population obtained by cell sorting.     

Mode of in vitro augmentation of natural killer cell activity by recombinant human interleukin 2: a comparative study of Leu-11+ and Leu-11- cell populations in cord blood and adult peripheral blood

Human newborn natural killer (NK) cell activity against K562 target cells was observed to be low compared with adult controls. Although Leu-7 (HNK-1)+ cells were negligible in cord blood, the proportions of Leu-11+ cells were equal to those of adult peripheral blood. Leu-11+ cells sorted from cord blood lymphocytes, as well as from adult lymphocytes exhibited the morphology of granular lymphocytes. In this study, we have investigated the phenotypic characterization of recombinant interleukin 2 (rIL 2)-induced cytotoxic lymphocytes against K562 cells by using anti-Leu-11 monoclonal antibody. Spontaneous cytotoxicity of lymphocytes was restricted to Leu-11+ cells in cord blood, as well as in adult blood, but this activity was low in cord blood Leu-11+ cells as compared with that of adult ones. NK cell activity of adult Leu-11+ cells could not be additionally enhanced after an 18-hr incubation with rIL 2(25 U/ml), whereas rIL 2 could potentiate the cytotoxicity of cord blood Leu-11+ cells approximately to the adult levels. It should be noted that cytotoxic activity of both Leu-11- cells from cord blood and adult blood that had no basal NK cells activity could be significantly potentiated by rIL 2. On the other hand, lymphokine-activated killer cells cytotoxic for HL-60 cell line could not be generated, and no proliferation of the lymphocytes was detected after an 18-hr incubation with rIL 2. It was shown that rIL 2 could not enhance the ability to bind to target cells in Leu-11+ and Leu-11- cells by means of a single cell conjugate assay, but the rate of target lysis of Leu-11+ cells from cord blood was significantly enhanced by rIL 2. These results suggested that rIL 2-induced cytotoxic effector cells were heterogeneous, and rIL 2 might potentiate the cytotoxicity of functionally immature NK cells or NK precursor cells.     

Antiviral effect of lymphokine-activated killer cells: characterization of effector cells mediating prophylaxis

Lymphokine-activated killer (LAK) cells generated by cultivation of C57BL/6 mouse spleen cells in the presence of recombinant interleukin-2 were transferred into natural killer (NK) cell-deficient suckling mouse recipients. These mice were then challenged with either murine cytomegalovirus (MCMV) or lymphocytic choriomeningitis (LCMV) and sacrificed 3 days later. No interleukin 2 infusions were given. Mice receiving as few as 5 x 10(5) LAK cells had several 100-fold decreases in spleen MCMV titers as compared with untreated mice. This treatment had no effect on spleen LCMV titers. The LAK cell cultures contained 10 to 17% NK 1.1+, 50 to 55% Lyt-2+, and 33 to 50% immunoglobulin D+ cells. Double fluorescence labeling and in vitro cytotoxicity assays with fluorescence-activated cell sorting revealed at least two mutually exclusive killer cell populations. NK 1.1+ LAK cells resembled freshly isolated activated NK cells with regard to target cell range (YAC-1 cell killing greater than L-929, P815, and EL-4 cell killing), large granular lymphocyte (LGL) morphology, and decreased ability to lyse interferon (IFN)-treated target cells. Lyt-2+ LAK cells lysed the targets mentioned above but at lower levels and without the differences in susceptibility mentioned above. These Lyt-2+ LAK cells also had a decreased ability to lyse IFN-treated targets, in contrast to classic cytotoxic T lymphocytes, which lyse IFN-treated targets far more efficiently than untreated targets. Purified populations of LAK cells obtained by fluorescence-activated cell sorting were used in the antiviral protection model. The results showed that protection against MCMV could be mediated by NK 1.1+, NK 1.1-, Lyt-2+, Lyt-2-, and IgD- populations but not by IgD+ cells. The five protective populations all had in common the LGL phenotype and cytotoxic activity in vitro. The IgD+ population did not contain LGLs, lyse target cells in vitro, or mediate an antiviral effect in vivo. These results suggest that LAK cells may be therapeutically useful against certain virus infections (MCMV) but not others (LCMV) and that despite their heterogeneity in antigenic phenotype and cytotoxic activity, their pattern of antiviral activity in vivo resembles that of NK cells, which protect against MCMV but not LCMV.     

Leu-Leu-OMe sensitivity of human activated killer cells: delineation of a distinct class of cytotoxic T lymphocytes capable of lysing tumor targets

Sensitivity to L-leucyl-L-leucine methyl ester (Leu-Leu-OMe) was used to characterize the phenotype of human activated killer cells. Natural killer cells (NK) and the precursors of both the alloantigen-specific cytotoxic T lymphocytes (CTL) and the NK-like activated killer cells generated after stimulation with allogeneic cells were deleted from human peripheral blood lymphocytes by preincubation with Leu-Leu-OMe. It was noted, however, that cytotoxic lymphocytes could be generated from Leu-Leu-OMe-treated lymphocyte precursors after 2 to 6 days of culture with the nonspecific mitogen, phytohemagglutinin (PHA). The characteristics of these killer cells indicated that they were a unique population that could be distinguished from other cytotoxic cells. Killing by these cells exhibited slow kinetics in that 18 hr cytotoxicity assays were required to detect full cytotoxic potential. When 18 hr assays were used, PHA-stimulated cytotoxic cells generated from Leu-Leu-OMe-treated lymphocytes were able to kill both NK-sensitive K562 cells and the relatively NK-resistant renal cell carcinoma cell line, Cur. These cytotoxic lymphocytes were HNK-1, Leu-11b (CD16), and OKM1 (CR3)-negative at both the precursor and effector stage of activation. Furthermore, these cells were derived from a CD3-positive precursor. Finally, killing by activated effectors was inhibited by OKT3. Unlike activation of Leu-Leu-OMe-sensitive large granular lymphocytes, generation of these cytotoxic T cells was totally prevented by treatment with mitomycin c before stimulation. Thus, a unique class of tumoricidal T cells can be characterized by resistance of lymphocyte precursors to a concentration of Leu-Leu-OMe, which has been shown to ablate NK, mixed lymphocyte culture-activated NK-like cytotoxic precursors, and the precursors of alloantigen-specific CTL.     

A human NK and K cell subset shares with cytotoxic T cells expression of the antigen recognized by antibody OKT8

The antigen recognized by monoclonal antibody OKT8 is expressed on the cell membrane of 30 to 50% of human NK/K cells. The reactivity of OKT8 with NK/K cells was determined by indirect methods (treatment of the effector cells with OKT8 antibody and complement (C) and separation of OKT8(+) and (-) effector cell populations by fluorescence-activated cell sorting or by rosetting techniques) and, at single cell level, by C-dependent lysis of effector NK cells that bind and kill K562 targets. Analysis by indirect immunofluorescence (flow cytofluorometry) of lymphocyte subpopulations mediating NK/K cytotoxic activity and deprived of OKT8(+) T cells reveals that the NK/K cell subset bears OKT8 antigen at a density lower than that present on cytotoxic T cells. The OKT8 antigen on NK/K cells is trypsin- and pronase-sensitive, but it is resynthesized by the same effector cells during 24 hr of culture at 37 degrees C. OKT8 antibody does not inhibit NK killing, and, on a per cell basis, OKT8(+) cells within the NK/K subset mediate the same level of cytotoxic activity as OKT8(-) NK/K cells. Analogous results were obtained by using anti-Leu-2a, an antibody with the same specificity as OKT8 on cytotoxic/suppressor T cells, but not when OKT5 was used, which might identify a distinct epitope on the same antigenic molecule. The possible significance of these findings in understanding the cell lineage of NK/K cells is discussed.     

Identification of effector cells for TNFalpha-mediated cytotoxicity against WEHI164S cells

WEHI164S cells were found to be very sensitive targets for in vitro killing in a 6-h culture when liver or splenic lymphocytes were used as effector cells in mice. Of particular interest, a limiting cell-dilution analysis showed that effector cells were present in the liver with a high frequency (1/4,300). In contrast to YAC-1 cells as NK targets, perforin-based cytotoxicity was not highly associated with WEHI164S killing. The major killer mechanism for WEHI164S targets was TNFalpha-mediated cytotoxicity. By cell sorting experiments, both NK cells and intermediate T cells (i.e., TCR(int) cells) were found to contain effector cells against WEHI164S cells. However, the killer mechanisms underlying these effector cells were different. Namely, NK cells killed WEHI164S cells by perforin-based cytotoxicity, TNFalpha-mediated cytotoxicity, Fas ligand cytotoxicity, and other mechanisms, whereas intermediate T cells did so mainly by TNFalpha-mediated cytotoxicity. These results suggest that TNFalpha-mediated cytotoxicity mediated by so-called natural cytotoxic (NC) cells comprised events which were performed by both NK and intermediate T cells using somewhat different killer mechanisms. Intermediate T cells which were present in the liver were able to produce TNFalpha if there was appropriate stimulation.     

Bacterial superantigens as anti-tumour agents: induction of tumour cytotoxicity in human lymphocytes by staphylococcal enterotoxin A

Summary Activation of lymphocytes by interleukin-2 (IL-2) induces lymphokine-activated killer (LAK) cells that show promising effects on tumour growth in clinical trials. We examined the effect of the superantigen staphylococcal enterotoxin A (SEA) on anti-tumour activity of freshly prepared human lymphocytes. Picomolar amounts of SEA rapidly induced cytotoxic activity against K562 and Raji cells as well as some natural-killer(NK)-resistant tumour cell lines. Cytotoxic activity was not dependent on target cell expression of either major histocompatibility complex (MHC) class I or II antigens as shown using mutated cell lines. Cell-sorting experiments showed that the activity was expressed by NK (CD5^–CD56⁺) as well as T (CD5⁺) cells, although the former contained the majority of cytotoxic activity. NK cells could not be directly activated by SEA. In contrast, SEA activated purified T cells to the same extent as in bulk cultures. It is suggested that SEA activation of NK cells is secondary to that brought about by lymphokines produced by T cells. Activation of LAK cells with SEA was comparable in magnitude as well as target cell spectrum to that of IL-2. In addition to the LAK-like cytotoxic activity induced by SEA, a superimposed cytotoxicity towards target cells expressing MHC class II antigens coated with SEA was observed. This staphylococcal-enterotoxin-dependent cell-mediated cytotoxicity (SDCC) was exclusively mediated by T cells. It is well established that MHC class II antigens function as receptors for staphylococcal enterotoxins on mammalian cells and that the complex between MHC class II antigen and — SEA apparently functions as a target structure for activated T cells with target cell lysis as a consequence. Activation of T lymphocytes with IL-2 also resulted in the capability to mediate SDCC. Staphylococcal enterotoxins represent a novel way of inducing anti-tumour activity in human lymphocytes, which could be of value in therapeutic applications.     

Suppression of human natural and antibody-dependent cytotoxicity by soluble factors from unstimulated normal lymphocytes

Serum-free culture supernatants of unstimulated normal human peripheral blood mononuclear cells contain soluble suppressor factor(s) (SSF) that significantly inhibit natural (NK) and antibody-dependent cellular cytotoxic (ADCC) activities of allogenic lymphocytes against a variety of target cells. Lymphocytes precultured with increasing concentrations of SSF showed a dose-dependent suppressive effect on these cytotoxic functions that was optimal at a concentration of 20% volume/volume. Adherent cells were not required for the production of SSF. Suppression was evident even at higher effector: target cell ratios and the inhibition was not reversed by washing lymphocytes. SSF was not itself cytotoxic, was stable at 56 degrees C, and its suppressive effect was maximal after 72 hr of incubation with effector lymphocytes. Initial estimate of the molecular weight of SSF by ultra-filtration was less than 20,000 daltons. Gel filtration of SSF on Sephacryl S-200 resulted in the elution of two peaks of activity; one in the region between markers of 13,700 and 25,000 daltons, and the other less than 13,700 daltons. Both fractions demonstrated significant suppressive activity on NK and ADCC functions of allogenic lymphocytes. SSF inhibition of NK activity could be partially reversed by incubating lymphocytes for 1 hr with human leukocyte interferon (IF) and almost completely reversed after 24 hr of IF treatment. A few selected monosaccharides (alpha-methyl-D-mannoside, L-fucose and L-rhamnose) showed a dose-dependent blocking effect on SSF activity, which suggests that SSF may act via receptor sites recognized by these sugars. As demonstrated for other lymphocyte functions, NK and ADCC activities may also be modulated by SSF elaborated by normal PBL.     

Cytotoxicity of human peripheral lymphocytes in cell-mediated lympholysis; antibody-dependent cell-mediated lympholysis and natural cytotoxicity assays after mixed lymphocyte culture.

Lymphocytes that have been purified by Ficoll-Hypaque centrifugation lose antibody-dependent and natural cytotoxic activities upon culture in tissue culture medium supplemented with human plasma. However, stimulation of peripheral lymphocytes in the mixed leukocyte culture (MLC) appears to enhance killer (K) and natural killer (NK) activities in addition to generating cytotoxic T ymphocytes. Enhancement of NK and antibody dependent activities appears to correlate with cell division as measured by 3H-thymidine uptake. However, elimination of dividing cells in the MLC by addition of 5-bromodeoxyuridine has no effect on NK and K cells activities. Since this treatment abolishes cell-mediated lympholysis mediated by cytotoxic T lymphocytes, it is a useful probe for determining the relative activities of NK, K, and cytotoxic T lymphocyte effector cells after lymphocyte stimulation.     

Sorting nexin 27 interacts with the Cytohesin associated scaffolding protein (CASP) in lymphocytes

CASP is a small cytokine-inducible protein, primarily expressed in hematopoetic cells, which associates with members of the Cytohesin/ARNO family of guanine nucleotide-exchange factors. Cytohesins activate ARFs, a group of GTPases involved in vesicular initiation. Functionally, CASP is an adaptor protein containing a PDZ domain, a coiled-coil, and a potential carboxy terminal PDZ-binding motif that we sought to characterize here. Using GST pulldowns and mass spectrometry we identified the novel interaction of CASP and sorting nexin 27 (SNX27). In lymphocytes, CASP's PDZ-binding motif interacts with the PDZ domain of SNX27. This protein is a unique member of the sorting nexin family of proteins, a group generally involved in the endocytic and intracellular sorting machinery. Endogenous SNX27 and CASP co-localize at the early endosomal compartment in lymphocytes and also in transfection studies. These results suggest that endosomal SNX27 may recruit CASP to orchestrate intracellular trafficking and/or signaling complexes.     

Analysis of the murine lymphokine-activated killer (LAK) cell phenomenon: dissection of effectors and progenitors into NK- and T-like cells

Murine as well as human lymphokine-activated killer (LAK) cells have been reported to have several characteristics of T lymphocytes and to be clearly distinct from natural killer (NK) cells. The present study of murine LAK cells showed that cytotoxic cells generated in the presence of interleukin 2 IL 2 were heterogeneous with respect to cell surface markers of progenitor as well as effector cells. Negative selection of cells with antibodies and complement or positive selection by fluorescence-activated cell sorting unequivocally showed that LAK effector cells consisted of at least two clearly distinct populations, the relative contribution of which was dependent on donor organ and target cells studied. Approximately 40% of the cytotoxic activity of spleen-derived effector cells active against the NK-resistant targets EL-4 or MCA-5 was eliminated by treatment with antibodies to the NK-markers asialo-GM1 and NK 1 (NK-LAK). Approximately 60% of cytotoxic activity was associated with cells expressing the T cell marker Lyt-2, lacked NK 1, and was lacking or expressed only small amounts asialo-GM1 (T-LAK). The NK-LAK cells were of greater importance for the cytotoxic activity against the standard NK target YAC-1, although T-LAK cells also excerted significant cytotoxicity against this cell line. Limiting dilution analysis estimated that the minimal frequency of precursors developing into cells with cytotoxic activity against EL-4 was 1/6700 in spleen and 1/4200 in peripheral blood. The frequency of cells developing into cytotoxic effectors against YAC-1 cells was 1/3700 and 1/1450 in spleen and peripheral blood, respectively. Depletion of progenitor cells from spleen or peripheral blood expressing NK 1 or Lyt-2 by treating the cells with antibodies to these structures and complement indicated that NK-1-expressing cells were the dominating progenitor of the LAK cells irrespective of target cells used. Culture of murine lymphoid cells from spleen or peripheral blood with high concentrations of IL 2 results in the emergence of two different killer cell populations with phenotypic similarities to NK and T cells, respectively, both being able to kill targets resistant to resting NK cells. In contrast to numerous earlier reports, we concluded that LAK cells are heterogeneous with respect to surface markers, with a major population of LAK cells apparently representing IL 2-activated cells expressing cell surface markers associated with NK cells.     

Human killer cells and natural killer cells: distinct subpopulations of Fc receptor-bearing lymphocytes

Unstimulated human peripheral blood lymphocytes were depleted of K cells, which mediate antibody-dependent cellular cytotoxicity (ADCC) without removing NK cells, which mediate natural killing (NK). K cell depletion was achieved by buoyant centrifugation removal of lymphocytes that bound to glutaraldehyde-treated P815-AB cells at high lymphocyte-to-target ratios. Likewise, NK cells were removed with glutaraldehyde-treated K562 cells without removing K cells. Furthermore, both cytotoxic cell populations were observed directly in one agarose single-cell cytotoxic assay (ASCA) using P815-AB and K562 cells simultaneously as target cells. Moreover, the percentage of total cytotoxic cells was equal to the sum of the percentage of K and NK cells observed in separate ASCA. Collectively, these results indicate that K cells and NK cells are distinct subsets of FcR-bearing lymphocytes. One subset, K cells, has more avid Fc receptors (fcR) than NK cells and are 'activated' via thier FcR to kill antibody-coated target cells. The second subset, NK cells, have less avid FcR and are not 'activated' through their FcR to kill antibody-coated target cells.     

Characterization of human large granular lymphocyte subpopulations: comparison of the phenotype of NK cells and of interleukin 2-dependent progenitors of cytolytic effector cells

Antigenically different subpopulations of human large granular lymphocytes (LGL) were identified according to their reactivity with monoclonal antibodies (MoAb). Antigen-positive and -negative subsets were isolated by immunoaffinity columns using a Sepharose 4B gel coupled with F(a')2 goat anti-mouse IgG or by flow cytometry cell sorting. The distinct LGL subsets were tested for natural killer (NK) activity against a panel of tumor targets: K562, Daudi, Alab; and for antibody-dependent cellular cytotoxicity (ADCC) against antibody-coated RL male 1 cells. LGL positively selected for any of the following phenotypic markers: B73.1+, OKM1+, OKT11+, and OKT10+ were highly cytotoxic, while B73.1- and OKM1- cells were completely devoid of NK activity. The OKT10- and OKT11- LGL subsets were occasionally cytotoxic, with low levels of reactivity. LGL subpopulations were also tested in a limiting dilution assay (LDA) for their capacity to proliferate in medium supplemented with interleukin 2 (IL-2) and to develop NK-like cytotoxic activity. The majority of proliferative progenitors have the following phenotype: OKT11+, OKM1-, B73.1-, and OKT10-, while the majority of progenitors for cytotoxic cells were OKT11+, OKM1+/-, OKT10+, and B73.1-. Results indicate that although B73.1+ cells can grow, the mature B73.1+ NK cells seem to be primarily derived in vitro from a small subset of less differentiated B73.1 pre-NK progenitors in the peripheral blood lymphocytes.     

Bacterial activation of human natural killer cells. Characteristics of the activation process and identification of the effector cell

We showed previously that contact of human peripheral blood lymphocytes with glutaraldehyde-fixed Salmonella bacteria augmented their cytotoxic capacity against NK-sensitive targets. We have now analyzed the characteristics of the activation and also identified the subsets of lymphocytes responding to bacterial contact. Blocking of protein synthesis with cyclohexamide totally abrogated bacterial induction of activated killing (AK), whereas inhibition of DNA synthesis with mitomycin C did not significantly affect the capacity of lymphocytes to respond to bacterial contact. Both the induction and the effector phase of AK were radioresistant. The AK cells exhibited efficient lytic activity, comparable to that induced by recombinant IL 2 (rIL 2), against NK-resistant targets (including both hematopoietic and solid tumor cell lines). All inducible cytotoxic activity was contained within the subset of lymphocytes expressing Leu-19 (NKH-1) antigen. Leu-19- lymphocytes exhibited no significant NK activity and could not be further stimulated by bacterial contact, rIL 2, or IFN-alpha. Within the Leu-19+ lymphocyte subset, two distinct cell types were present; CD3-, Leu-19+ NK cells and CD3+. Leu-19+ T cells. The CD3+, Leu-19+, T cells mediated low levels of non-MHC-restricted cytotoxicity against K562, but did not respond to bacterial contact, even though rIL 2 could augment their lytic activity slightly. However, the cytotoxic activity of CD3-, Leu-19+ NK cells was significantly augmented by bacterial contact. Within the CD3-, Leu-19+ NK cell population both CD16+ and CD16- cells responded to bacterial activation. The CD3-, CD16-, Leu-19+ cells constituted 1 to 4% of the Percoll-fractionated low buoyant density lymphocytes and accounted for the activation seen within the CD16- lymphocyte population. Thus bacterial stimulation of NK activity seems to be mediated for the most part via CD16+, Leu-19+ cells, and a minor overall contribution is mediated via CD3-, CD16-, Leu-19+ cells. No apparent involvement of T cells was seen in the lytic response of lymphocytes to bacterial contact.     

A novel negative regulator molecule, Cho-1, is involved in the cytotoxicity by human natural killer cells but not in cytotoxic T lymphocytes.

We previously reported the cytotoxic negative regulatory molecule, Cho-1, that was expressed on the cell surface of rat fetal fibroblast cells in the cytotoxicity by natural killer (NK) cells. This molecule was IFN-gamma-inducible, but appeared to be different from MHC class I. It was expressed on NK-resistant cells but not on NK-sensitive murine target cells such as YAC-1. In this paper, first we determined whether Cho-1 could also act as the negative regulatory molecule in a human NK-resistant HEPM line. Our data strongly suggested that Cho-1 could act as such a negative regulatory molecule in human NK cytotoxicity. The immunoprecipitates made with HEPM cell lysate and anti-MHC class I monoclonal antibody (mAb) did not react against anti-Cho-1 mAb, indicating that Cho-I was different from MHC class I. Second, an assessment was made as to whether or not this molecule is involved in the cytotoxicity of CD8 (+) cytotoxic T lymphocytes (CTL) against human autologous tumor cells. The data indicated that although this cell surface molecule was expressed on certain tumor lines, it was not involved in the cytotoxic mechanism of CTL. Thus, Cho-1 appeared to be the novel regulatory molecule in the NK cytotoxic mechanism.     

Inhibition of in vitro granulopoiesis by autologous allogeneic human NK cells

This study demonstrates the ability of human NK cells to inhibit in vitro granulopoiesis of autologous and allogeneic BM cells. NK lytic activity and GM-CFC inhibition was present among nonstimulated lymphocytes from healthy donors and could be increased by treatment of PBL with IFN. Both the cytotoxic NK cells and the GM-CFC inhibitory cells could be enriched for among nonadherent, low-density cells. High-density cells were not cytotoxic, only inhibitory to a small extent, and could become neither cytotoxic nor more inhibitory after IFN treatment. In contrast, low-density cells showed an increased cytotoxic and GM-CFC inhibitory capacity after IFN treatment. The NK mediated GM-CFC inhibition was dependent on cell contact with BM cells, increased with longer preincubation times, and was most efficient against 7-day GM-CFC as compared with 14 day GM-CFC progenitors. In conclusion, these data provide new information about the human NK cell as a potent inhibitor of in vitro granulopoiesis and also as a possible regulator of hematopoiesis in vivo.