Characteristics of natural killer cells (NK cells) and features of the cytotoxic test in Syrian hamsters

The cytotoxic test in vitro with the use of xenogeneic target cells of human myeloma, strain K-562, labeled with 51Cr has demonstrated natural cytotoxicity of lymphoid cells from noninbred Syrian hamsters. This cytotoxicity occurs at the cost of non-adherent splenocytes. NK may be isolated over the gradient density of ficoll (1.078), selective for large granular lymphocytes. To detect the maximal lytic activity of NK from Syrian hamsters in the cytotoxic test in vitro, they should be brought into 10-12 hour contact with sensitive target cells K-562. In Syrian hamsters, the highest natural cytotoxicity is shown by the cells of the blood and spleen. In the bone marrow and thymus, it is little pronounced and is virtually absent from the peripheral lymph nodes.     

Natural cytotoxic T cells (NCTC) that differ from natural killer (NK) and natural cytotoxic (NC) cells are present in Peyer's patches of mice

We have examined noninduced cytotoxicity of mouse gut associated and peripheral lymphoid tissues for a wide variety of syngeneic as well as allogeneic cell lines and lymphoblasts. Lymphoid cells from Peyer's patches were found to lyse these targets in a 3-hr chromium release assay whereas lymphoid cells from intestinal mucosa, mesenteric or peripheral lymph nodes, spleen, and thymus did not. The variety of targets toward which Peyer's patch cells were cytotoxic established the latter as nonspecific and H-2 unrestricted. The cell responsible for the lytic event was identified as possessing Thy 1.2 and Ia surface antigens. This naturally cytotoxic T cell (NCTC) was found to be adherent to nylon-wool but not to plastic plates. Although both natural killer cell (NK) and non-NK targets served as targets for the NCTC, the latter were further differentiable from NK cells by lack of asialo GM1 surface marker, which is present on NK cells. In addition, NCTC remained fully functional in mice given either of the drugs cyclophosphamide or cortisone. Each of these drugs, in the doses used, markedly reduced poly(I:C)-induced NK activity. Thus, NCTC differs from NK on the basis of the spectrum of targets against which it is functional, phenotypic surface markers, insusceptibility to stimulation with poly(I:C), and insensitivity to diminution by the immunosuppressive drugs cyclophosphamide and hydrocortisone. Since NCTC is a Thy 1.2 antigen-bearing cell and is detectable in a 3-hr cytotoxic assay, it also differs from the natural cytotoxic (NC) cell. NC lacks the Thy 1.2 marker and becomes detectable only in an 18-hr cytotoxic assay. Thus, NCTC is neither an NK nor an NC cell. We have discussed the possibility that the three naturally occurring cells may be related by being dedifferentiated descendants of an antigen-specific cytotoxic T lymphocyte (CTL). Alternatively, since NCTC is confined to an anatomical site prone to ample antigenic exposure and is still identifiable as a T cell, it may be in linear transition from the CTL to the NK or NC stages.     

Effect of human recombinant interferon on cytotoxic activity of natural killer (NK) cells and monocytes

Studies have been performed on the in vitro immunologic effects of homogeneous recombinant human leukocyte interferon, IFLrA. Large granular lymphocytes, enriched for natural killer (NK) cell activity, were pretreated wtih IFLrA or natural interferon preparations and then tested for augmentation of NK activity and of antibody-dependent cell-mediated cytoxicity (ADCC). Monocytes were tested for cytolytic and cytostatic activity in 48–72 hr radioisotopic assays performed in the presence or absence of interferon. Treatment with IFLrA caused significant augmentation of NK, ADCC, and monocyte-mediated cytotoxic activities. Even 10 units of IFLrA induced augmentation of NK activity, and 100 units or more boosted monocyte-mediated activity. The effects in each of these assays were species-specific, with no detectable effects on the activity of mouse effector cells. These results indicate that homogeneous recombinant interferon has potent in vitro immunomodulating effects and thus provide a basis for carefully examining the in vivo effects of this protein on host defenses in forthcoming clinical trials with cancer patients.     

Redistribution of natural killer (NK) cell frequency and NK cytotoxic activity in primary IgA nephropathy.

Recent findings have indicated an imbalance of immune responsiveness in primary IgA nephropathy (IgAN). Thus natural killer (NK) cell frequency and NK cytotoxicity were evaluated in fifteen IgAN patients. CD8+, CD11+, CD56+ and CD57+ lymphocyte percentages in IgAN individuals fell within normal values, while a significant decrease of CD16+ cells was observed in the same group of patients. In contrast, NK activity overlapped that seen in controls as assessed by an agarose-single cell cytotoxic assay. To further investigate the discrepancy between CD16+ cell level and NK cytotoxic activity in IgAN, the proportion of CD11+ CD57+, CD56+ CD16+ and CD57+ CD16+ lymphocytes was determined. In spite of the unaffected CD56+ CD16+ cell frequency, IgAN subjects exhibited a significant decrease of CD11+ CD57+ and CD57+ CD16+ lymphocyte percentages in comparison to controls. It is suggested that a redistribution of NK lymphocyte subsets occurs in IgAN. This may have an important role in the impairment of the immunoregulatory network.     

In vivo bidirectional regulation of mouse natural killer (NK) cell cytotoxic activities by Leu-enkephalin: reversibility by naloxone.

Intraperitoneal injection of Leu-enkephalin (LENK, 10 or 7.5 mg/kg) induced bidirectional modulation of natural cytotoxic activities in spleens of CBA mice (suppression followed by enhancement). NK-cytotoxic activity was more affected than the ADCC. Early suppression of NK activity could be reversed by 4 x M excess of naloxone injected 20 min before LENK, suggesting that the suppression was mediated by opioid receptors. Subsequent increase of NK activity could not be abrogated by naloxone, at least not completely. Naloxone itself decreased NK activity 12 hours after treatment, but enhanced ADCC at 24 and 48 hours. This increase was abrogated by LENK. In addition to functional alterations, LENK also induced phenotypic changes of spleen cells, i.e. a decrease in the percentage of asialo-GM-1+ cells 24 hours posttreatment. There was no correlation between LENK-induced alterations of cytotoxic function and the percentage of cells with NK phenotype (GM-1+). Thus, LENK modulates cytolytic functions and the phenotype of NK cells in vivo in a complex way, which besides opioid mechanisms may also include non-opioid ones.     

Establishment of hybridoma cells with natural killer(NK)-like activity against syngenic tumor cells.

The 4D1D4 hybridoma cells were derived from the fusion of spleen cells from BALB/c nude mice with NS-1 mouse myeloma cells. The surface phenotypes of 4D1D4 hybridoma cells were Thy-1.2+, L3T4 (CD4)-, Lyt-2 (CD8)-, Asialo GM1+ and p-55 interleukin-2 (IL-2) receptor (CD25)-. This phenotypic pattern was consistent with the surface phenotype of NK cells. The 4D1D4 cells showed the definite killer activity against a syngenic tumor cell line, RL male-1, but not against an allogenic YAC-1 line. The killer activity of the 4D1D4 cells was not affected by the addition of exogenous IL-2. It was, therefore, suggested that 4D1D4 cells might be representative of resting NK cells with expression of no functional IL-2 receptors. The hybridoma technology might be useful for establishment of the cloned NK cells.     

Separation and isolation of rat natural killer (NK) cells from T cells with monoclonal antibodies

It has been difficult to clearly differentiate rat NK cells from cytotoxic T cells. In this study we have shown that rat NK cells do not express the T cell protein defined by the OX 19 antibody. By using the FACS we have isolated the OX 19- OX 8+ lymphocyte subset that contains virtually all the NK activity. The simultaneous use of the OX 19 and OX 8 antibodies allows the separation of NK cells from T cells.     

Demonstration of the antiviral role of natural killer cells in vivo with a natural killer cell-specific monoclonal antibody (NK 1.1)

A monoclonal antibody (NK 1.1) to mouse natural killer (NK) cells selectively depleted NK cell activity in virus-infected mice without significantly depressing other immune functions, including the development of virus-specific cytotoxic T cells. NK cell depletion with this antibody resulted in markedly enhanced plaque-forming unit titers of some (murine cytomegalo, Pichinde) but not other (mouse hepatitis, lymphocytic choriomeningitis) viruses. This confirms that NK cells do play a role in regulating certain infections and shows that this antibody provides a convenient tool for examining the role of NK cells in viral infections.     

A subpopulation of allospecific cytotoxic T-cell precursors with phenotypic characteristics of natural killer cells

We have proposed that natural killer (NK) cells are germ-line V-gene encoded prothymocytes specific for either self or non-self histocompatibility antigens. This hypothesis predicts that at least some precursors of allospecific cytotoxic T cells (allo-CTL) are NK cells. To test this we examined the effect of depleting NK cells and/or T cells (by complement lysis with anti-asialo GM1 and/or anti-Thy 1) on the development of allo-CTL induced during mixed lymphocyte culture (MLC). Removal of Thy 1+ cells from MLC responder populations prevented development of allo-CTL. This was partially reversed by addition of concanavalin A-conditioned medium (Con A-CM) to the MLC at day 0. Removal of asialo GM1+ cells eliminated NK activity measured at day 0, but failed to prevent development of allo-CTL of otherwise intact responder cells. However, removal of asialo GM1+ cells did prevent the Con A-CM dependent development of allo-CTL by responder cells depleted of Thy 1+ cells. These findings indicate that a subpopulation of allo-CTL precursors has the phenotypic characteristics of NK cells: absence or low density of Thy 1, and susceptibility to complement lysis by anti-asialo GM1.     

Suppression of natural killer (NK) cell activity of spleen cells by thymocytes

The in vitro influence of thymus cells on natural killer cell activity of spleen cells against prelabeled target cells (YAC-I and RL♂I) has been studied in syngeneic as well as in allogeneic murine models. In mixing experiments to demonstrate suppression, total thymocytes have been found to have no effect on NK activity of syngeneic or allogeneic spleen cells. Among several thymocyte fractions separated by velocity sedimentation, a relatively faster sedimenting fraction showed remarkable suppression of NK activity by spleen cells against two target cells. The suppressive effect of this particular fraction on NK activity was demonstrated to be proportional to the cell dose. The suppressive function was resistant to irradiation at 1000 or 2000 rad administered in vitro and was not restricted by the major histocompatibility complex. Moreover, the thymocyte fraction which induced suppression was not sensitive to NK-mediated cytolysi? by syngeneic spleen cells. The suppression of NK cytolysis in vitro by certain subpopulations of thymocytes as observed in the present studies may be consistent with a role for the thymus in regulating NK activity in vivo.     

Epinephrine-induced mobilization of natural killer (NK) cells and NK-like T cells in HIV-infected patients

HIV infection is known to cause changes in phenotype and function of natural killer (NK) cells. The aim of this study was to characterize the NK cells mobilized from peripheral reservoirs in human immunodeficiency virus (HIV)-infected patients and controls. Seventeen HIV-infected patients and eight age- and sex-matched controls received a 1-h epinephrine infusion. Epinephrine induced mobilization of high numbers of NK-like T cells with no difference between HIV-infected patients and controls. Interestingly, all subjects mobilized NK cells containing increased proportions of perforin, in particular the CD3(-)CD16(+)CD56(+) NK cell subset. The HIV-infected patients mobilized CD3(-)CD16(-)CD56(+) and CD3(-)CD16(+)CD56(+) NK cells to a lesser extent than did controls. In contrast, the HIV-infected patients mobilized relatively more CD3(-)CD16(+)CD56(-) NK cells independent of antiretroviral treatment. It is suggested that these cells represent an immature NK cell subpopulation possibly resulting from an impaired cytokine tissue environment in HIV-infected patients.     

Interleukin-10 reduces natural killer (NK) sensitivity of tumor cells by downregulating NK target structure expression

We examined the effect of exogenous IL-10 on the sensitivity of rat W14 and W31 tumor cells to natural killer (NK) cell-mediated cytotoxicity in relation to previously identified NK target structure (NKTS) expressed on these cells. We also examined the effect of endogenous interleukin-10 (IL-10) on rat IL-10 cDNA-introduced W31 cells (W31T-H, a high-IL-10-producer clone; W31T-L, a low-IL-10-producer clone). Both exogenous and endogenous IL-10 had no effect on the proliferative activity of these cells, but incubation of cells with recombinant human (rh) IL-10 resulted in a dose-dependent decrease in the expression of NKTS recognized by mAb 109. The expression level of NKTS on W31T-H cells was dramatically decreased compared with that on W31T-L cells and parental W31 cells. In addition, treatment of W31 cells with the culture supernatants of W31T-H cells could downregulate the expression of the NKTS. Moreover, NK sensitivity of W31T-H was suppressed down to a level equivalent to that of W31 cells pretreated with exogenous rhIL-10, and cytolysis could no longer be inhibited by mAb 109. We previously demonstrated that IL-10 downregulated MHC class I expression in this model. Nevertheless, NK susceptibility was also decreased. Taken together, these results suggest that the IL-10-mediated decrease in NKTS expression has a larger effect than decreased MHC class I expression on NK sensitivity. Thus, our data reveal a novel mechanism for an IL-10-mediated escape of tumor cells from host immune surveillance by downregulation of NKTS expression.     

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.     

Studies on murine natural killer (NK) cells. V. Genetic analysis of NK cell markers

This paper attempts to clarify the number and nomenclature of murine natural killer (NK) cell specific alloantigens by defining the genetic relationships between them, that is, are they coded by loci which are independent, allelic, or linked. Strain typing and F2 analyses using five alloantisera (C3H X BALB/c)F1 anti-CE, CE anti-CBA, NZB anti-BALB/c, C3H anti-ST, and BALB/c anti-DBA/2 revealed that (a) the alloantigens NK-1.1 and NK-3.1 are determined by distinct loci which are linked on the same chromosomes, (b) the alloantigen NK-2.1 is determined by an independently segregating locus to those coding for NK-1.1 and NK-3.1, (c) the alloantisera, CE anti-CBA and NZB anti-BALB/c, which have been designated anti-NK-2.1 alloantisera recognize different alloantigens coded by independent genetic loci. Thus, these five alloantisera detect four NK cell specific alloantigens which, based on the chronology of their discovery, have been designated NK-1.1-(C3H X BALB/c)F1 anti-CE, NK-2.1-CE anti-CBA, NK-3.1-C3H anti-ST, and BALB/c anti-DBA/2 and NK-4.1-NZB anti-BALB/c.     

Activation of natural killer (NK) cells in vivo with H-2 and non-H-2 alloantigens

Intraperitoneal inoculation of allogeneic lymphoid cells rapidly activates cytotoxic cells in the peritoneum which are nonadherent and express the NK-1, asialo-GM₁, and Thy-1 antigens. Allogeneic spleen cells were very efficient at activating these natural killer (NK) cells, while allogeneic thymocytes were much less effective. Heat-killed allogeneic cells or sonicates also could augment NK activity. — Incompatibility atH-2K, H-2I-A, orH- 2D readily evoked NK cell activity, whileH-2S- andH-2I-E/C-associated disparities did not. Non-H- 2 differences also stimulated NK activity and augmentation was particularly evident inMls-disparate combinations. Thus, the same alloantigens which efficiently activate T cells also activate NK cells.     

Activation of natural killer (NK) T cells during murine cytomegalovirus infection enhances the antiviral response mediated by NK cells

NK1.1+ T (NKT) cells are efficient regulators of early host responses which have been shown to play a role in tumor surveillance. The relevance of NKT cells in immune surveillance of viral infections, however, is not well understood. In this study, we investigated the functional relevance of NKT cells in controlling herpesvirus infections by using challenge with murine cytomegalovirus (MCMV) as the study model. This model has proven to be one of the best systems for evaluating the role of NK cells during virus infection. Using gene-targeted mice and alpha-galactosylceramide (alpha-GalCer) as an exogenous stimulator of NKT cells, we have analyzed the role of these cells in the immune surveillance of MCMV infection. Our studies in NKT-cell-deficient, T-cell receptor Jalpha281 gene-targeted mice have established that classical NKT cells do not play a critical role in the early clearance of MCMV infection. Importantly, however, activation of NKT cells by alpha-GalCer resulted in reduced viral replication in visceral organs. Depletion studies, coupled with analysis of gene-targeted mice lacking perforin and gamma interferon (IFN-gamma), have revealed that the antiviral effects of alpha-GalCer involve NK cells and have clearly demonstrated that the antiviral activity of alpha-GalCer, unlike the antitumor one, is critically dependent on both perforin and IFN-gamma.     

Inability of interferon to protect virus-infected cells against lysis by natural killer (NK) cells correlates with NK cell-mediated antiviral effects in vivo

Murine cytomegalovirus (MCMV) is a natural killer (NK) cell-sensitive virus, whereas lymphocytic choriomeningitis virus (LCMV) is an NK cell-resistant virus. Selective depletion of NK cell activity by injection of mice with anti-asialo GM1 antibody enhanced synthesis of MCMV but not that of LCMV when mice were simultaneously infected with the two viruses. This suggests that the NK cell-mediated antiviral effects may depend on target cell susceptibility to NK cell-mediated lysis rather than the ability of a virus to induce a specialized antiviral NK cell. In support of this concept, activated NK cells isolated from either MCMV- or LCMV-infected mice had similar patterns of killing against all targets tested. Mouse embryonic fibroblasts (MEF) infected with MCMV were less sensitive to lysis by activated NK cells than either uninfected or LCMV-infected MEF. However, when MEF were pretreated with IFN, activated NK cell-mediated lysis against MCMV-infected MEF was undiminished and was much higher (up to fourfold) than that against uninfected MEF, whose sensitivity to lysis was almost totally abolished by IFN pretreatment. LCMV-infected MEF were also protected by IFN against activated NK cell-mediated lysis. During infection, the virus-induced IFN may protect uninfected and LCMV-infected cells from IFN-activated, NK cell-mediated lysis, but MCMV-infected cells may remain sensitive to lysis. This could explain how NK cells play a role in resistance to MCMV but not LCMV.