Natural Killer Cytotoxicity and Antibody-Dependent Cell Cytotoxicity to Herpes Simplex Virus-Infected Target Cells in Murine Pregnancy

ABSTRACT: In vitro natural killer cytotoxicity (NKC) and antibody-dependent cell cytotoxicity (ADCC) activity against herpes simplex virus (HSV)-infected cells were evaluated in a pregnant murine model (C₅₇B1₆inbred strain). Virgin (n = 16) and pregnant (late gestation) mice (n = 15) were infected intraperitoneally with HSV, type 1. After 18 hr, a 0.5-ml aliquot of the peritoneal wash was frozen for virus plaque assay, and the cells were cultured in the ⁵¹chromium release assay for NKC and ADCC. %NKC (mean ± S.E.) to HSV-infected targets was significantly suppressed (P < 0.05) in pregnant mice, 10.3% ± 1.9, compared to that of virgin mice, 32.5% ± 2.5. This suppression was abrogated with HSV-specific antisera (%ADCC); 53.9% ± 4.4 (pregnant) compared to 49.1% ± 3.6 (virgin). The diminished NKC activity in pregnant mice was reflected in an increased mean number of virus particles in the peritoneal wash, 266 + 66 PFU/ml, compared to 38 ± 11 PFU/ml in virgin mice (P < 0.05). We concluded that NKC, but not ADCC, to HSV-infected targets was suppressed and that HSV elimination was impaired in pregnant mice.     

The Effect of Tunicamycin on Target Cell Susceptibility to Natural Killer Cell Cytotoxicity

Several sets of data indicate the possibility that carbohydrate moieties on the target cell are important structures in natural killer (NK) cell-mediated lysis. Striking changes in the NK susceptibility of targets can be induced in several systems involving in vitro differentiation of tumour cell lines. The effect on target cells of the glycosylation inhibitor tunicamycin, which acts by blocking the dolichol-dependent asparagine-linked glycosylation pathway was investigated. Using several different tumour cell lines we can conclude that: asparagine-linked carbohydrate chains do not contribute directly to NK susceptibility, induced differentiation may or may not be linked with a change in NK susceptibility, and secondary changes caused by tunicamycin treatment may lead to alterations in the gangliosides, a finding that is positively correlated with decreased NK susceptibility.     

High Dose IL-2-Activated Murine Natural Killer (A-NK) Cells Accumulate Glycogen and Granules, Lose Cytotoxicity, and Alter Target Cell Interaction In Vitro

Activated natural killer (A-NK) cells, defined by immunophenotype and selected by adherence to the plastic, were cultured from murine splenocytes for up to 10 days with the addition of 1000 U/ml of recombinant human IL-2 at 48 h intervals. During culture days 2–4 with high DNA synthesis the initially non-granulated small cells established large granular lymphocyte (LGL) morphology and then differentiated further into giant hypergranulated cells with huge accumulations of glycogen. Timed EM observations indicated that specific dual-compartment (lytic) granules arose by a sequence of events starting with neo-synthesis of small progenitors with a dense core and a few membranous lamellae at one pole. Core and vesicular regions probably expanded independently to give the mature organization of the granule. Eventually, the vesicular region of granules contained large amounts of multi-lamellar material and probable debris, and the dense core could be multiplied. Intracellular proteoglycans, visualized with Cupromeronic Blue cytochemistry, were organized in a three-dimensional network within the dense cores. In contrast with earlier reports, and in spite of several-fold increased granularity, the in vitro cytotoxicity of the A-NK cells against YAC-1 and B16 cells decreased after the third day of culture. A-NK cells with glycogen accumulations caused focal clearing in melanoma monolayers whereas younger effectors adhered to the targets. It is concluded that high dose IL-2 stimulation causes more far-going progressive morphological and functional differentiations of the A-NK cells than has previously been observed with bearing for the use of these cells in experimental adoptive immunotherapy.     

Surface Ly-5 glycoprotein in murine natural killer (NK) cell development, target binding and cytotoxicity

The role of surface Ly-5 glycoprotein expression in the binding and lysis of susceptible tumor targets by natural killer cells was studied using NK cell-enriched splenocytes from 6-8 week old C57BL/6 mice which were reacted with anti-Ly-5 serum in the presence or absence of a source of complement. A conjugate assay was used to demonstrate that abrogation of tumor cell lysis by anti-Ly-5 serum involved the inhibition of NK cell binding to susceptible YAC-1 targets. Additionally, reconstituted membrane vesicles from NK cell-enriched splenocyte populations blocked binding of effector cells to YAC-1 lymphoma targets, a phenomenon which was abrogated by pretreatment of vesicles with anti-Ly-5 serum. Indirect immunofluorescent labeling and cell sorting were used in the physical separation of Ly-5+ and Ly-5- cells to examine the effect of interferon and interleukin preparations on Ly-5 expression and Nk activity. Three hour treatment of sorted Ly-5- cells with murine alpha + beta interferon resulted in conversion of 22% of the cells to an Ly-5+ phenotype, as well as a significant increase in the percent specific lysis of NK-susceptible YAC-1 targets when compared to freshly sorted Ly-5- cells (29.5 +/- 1.9 vs 2.6 +/- 4.0; p less than .001). In vitro proliferation of sorted Ly-5- cells was induced by three week culture in an interferon- and interleukin-containing supernatant from ConA stimulated BALB/c splenocytes (CM), followed by repeat analysis of Ly-5 expression and cytotoxic activity. Cell sorter purified Ly-5- cells cultured in CM acquired substantial surface Ly-5 with concomitant high levels of cytotoxic activity that remained partially susceptible to inhibition by anti-Ly-5 serum. The data presented suggest that surface Ly-5 glycoprotein expression is important for binding of freshly isolated NK cells to YAC-1 targets. In addition, Ly-5- precursors of NK cells are present in murine splenic tissues and can be induced by CM to become highly active effector cells with increased surface Ly-5 expression. The persistent susceptibility of a subset of these cells to inhibition of cytotoxic activity by anti-Ly-5 serum provides additional evidence of an important role for the Ly-5 glycoprotein in the natural killer cell cytolytic mechanism against certain targets.     

Activating Natural Killer Cell Receptors,Selectins, and Inhibitory Siglecs Recognize Ebolavirus Glycoprotein

Expression of the extensively glycosylated Ebolavirus glycoprotein (EBOV-GP) induces physical alterations of surface molecules and plays a crucial role in viral pathogenicity. Here we investigate the interactions of EBOV-GP with host surface molecules using purified EBOV-GP, EBOV-GP-transfected cell lines, and EBOV-GP-pseudotyped lentiviral particles. Subsequently, we wanted to examine which receptors are involved in this recognition by binding studies to cells transfected with the EBOV-GP as well as to recombinant soluble EBOV-GP. As the viral components can also bind to inhibitory receptors of immune cells (e.g., Siglecs, TIM-1), they can even suppress the activity of immune effector cells. Our data show that natural killer (NK) cell receptors NKp44 and NKp46, selectins (CD62E/P/L), the host factors DC-SIGNR/DC-SIGN, and inhibitory Siglecs function as receptors for EBOV-GP. Our results show also moderate to strong avidity of homing receptors (P-, L-, and E-selectin) and DC-SIGNR/DC-SIGN to purified EBOV-GP, to cells transfected with EBOV-GP, as well as to the envelope of a pseudotyped lentiviral vector carrying the EBOV-GP. The concomitant activation and inhibition of the immune system exemplifies the evolutionary antagonism between the immune system and pathogens. Altogether these interactions with activating and inhibitory receptors result in a reduced NK cell-mediated lysis of EBOV-GP-expressing cells. Modulation of these interactions may provide new strategies for treating infections caused by this virus.     

Effects of X-RAY Irradiation on Natural Killer (NK) Cell System. II. Increased Sensitivity to Natural Killer Cytotoxic Factor (NKCF)

Irradiation with low-doses of X-rays of tumor cells elevated their susceptibility to lysis by natural killer (NK) cells in an accompanying paper. Cytotoxicity assays conducted at the single cell level revealed that X-ray irradiation of K562 cells did not affect the number of effector-target conjugates but increased the frequency of dead conjugated target cells. During interaction with K562 cells large granular lymphocytes released a soluble cytotoxic factor (NKCF) that killed the target cells. X-ray irradiation did not affect the NKCF stimulatory ability of K562 cells, while it elevated their sensitivity to the lytic effect of NKCF. In contrast to X-rays, exposure to ultraviolet (UV) radiation of K562 cells did not elevate their NK sensitivity but rather reduced it. Treatment with mitomycin C produced no effect on NK sensitivity. These results indicate that X-ray irradiation elevates the target sensitivity to NKCF, which may be involved in the increased NK sensitivity, and that the X-ray effect may be different from that of UV radiation or DNA synthesis inhibition.     

Effects of X-RAY Irradiation on Natural Killer (NK) Cell System. II. Increased Sensitivity to Natural Killer Cytotoxic Factor (NKCF)

Abstract

Irradiation with low-doses of X-rays of tumor cells elevated their susceptibility to lysis by natural killer (NK) cells in an accompanying paper. Cytotoxicity assays conducted at the single cell level revealed that X-ray irradiation of K562 cells did not affect the number of effector-target conjugates but increased the frequency of dead conjugated target cells. During interaction with K562 cells large granular lymphocytes released a soluble cytotoxic factor (NKCF) that killed the target cells. X-ray irradiation did not affect the NKCF stimulatory ability of K562 cells, while it elevated their sensitivity to the lytic effect of NKCF. In contrast to X-rays, exposure to ultraviolet (UV) radiation of K562 cells did not elevate their NK sensitivity but rather reduced it. Treatment with mitomycin C produced no effect on NK sensitivity. These results indicate that X-ray irradiation elevates the target sensitivity to NKCF, which may be involved in the increased NK sensitivity, and that the X-ray effect may be different from that of UV radiation or DNA synthesis inhibition.     

Cell Surface Thiols, but not Intracellular Glutathione, are Essential for Cytolysis by a Cloned Murine Natural Killer Cell Line

Cell surface thiols are required for a line of cloned murine natural killer lymphocytes to bind to and lyse tumor target cells. These lymphocytes neither bound to nor killed YAC-1 or G₁T_c cells when the effector lymphocyte cell surface thiols were covalently coupled with the non-penetrating reagent, monobromotrimethylammoniobimane (qBBr). A limited number of thiol-bearing proteins were identified by gel electrophoresis on the cell surface using the fluorescence of the group that remains associated with the sulfur molecule. These results indicate that either one or more of these reactive proteins or different cell surface thiol-bearing molecules present at low frequencies are crucial to lymphocyte binding and killing. In contrast, we found little evidence that intracellular thiols are required for natural killer cell activity. Killing was relatively unimpaired when over 90% of lymphocyte glutathione was depleted with DL buthionine-S, R-Sulfoximine (BSO). Blocking the intracellular or the extracellular thiols of tumor targets had no effect on their ability to be lysed. Based on these data, we suggest that infrequently expressed extracellular thiols are required either for the conformation or for the disulfide crosslinking of proteins that participate in lymphocyte-mediated cytolysis.     

Natural Killer Cell Cytotoxicity and Antibody-Dependent Cellular Cytotoxicity to Herpes Simplex Virus-Infected Cells in Human Pregnancy

ABSTRACT: Natural killer cell (NKC) cytotoxicity and antibody-dependent cellular cytotoxicity (ADCC) represent the ability of human leukocyte effector cells to destroy target cells in the absence and presence of antibody, respectively. Since these immune systems play a pivotal role in the body's primary lines of defense against a variety of pathogens including herpes simplex virus (HSV), a study was undertaken to evaluate the influence of pregnancy on these systems. Eleven uncomplicated gravidas were followed serially through each trimester and compared to 11 nonpregnant female controls. Mononuclear cells were acquired by Ficoll-Hypaque centrifugation of heparinized blood. Chang liver cells infected with HSV-I were utilized as target cells in a ⁵¹Cr release assay. Mean NKC values in the pregnant patients were uniformly lower than in the controls. No similar decreases in ADCC activity were observed in a comparison between the two study populations. These data support previous observations suggesting that pregnancy represents a relatively immunocompromised state. Differences apparently exist between NKC and ADCC effector cell populations with regard to the influence of pregnancy. Although these physiologic alterations in immunoregulation may help support the fetoplacental allograph, detrimental conditions may exist regarding susceptibility to various pathogens such as HSV.     

ORIGINAL ARTICLE: Suppression of Natural Killer Cell Cytotoxicity in Postpartum Women

Citation Groer M, El‐Badri N, Djeu J, Harrington M, Van Eepoel J. Suppression of natural killer cell cytotoxicity in postpartum women. Am J Reprod Immunol 2010; 63: 209–213 Problem Natural Killer (NK) cell numbers and cytotoxicity are suppressed during pregnancy. Little is known about postpartum NK number and function. Method of study Postpartum women (n = 39) were studied at one week and then monthly over the first six postpartum months. The standard natural killer cell cytotoxicity assay (NKCA) was performed. This is a Cr51 release assay from K562 cells cultured with peripheral blood mononuclear cells (PBMCs). Results Data indicate suppression of NK cytotoxicity in postpartum women. Cytotoxicity at each effector:target (E:T) ratio showed a drop from 1 week postpartum, reaching a nadir at around 2 months, and a trend towards recovery of cytotoxicity from 3 to 6 months. Lytic units (LUs) from pre‐incubated cells from postpartum women were lower than age‐matched, non‐pregnant, non‐postpartum controls through the fifth postpartum month. Conclusion These data suggest that the postpartum period, like pregnancy, is characterized by decreased NK cytotoxicity activity. This suppressed NK cytotoxic effect may result as a response to interaction with tolerized fetal microchimeric cells accumulated during pregnancy in maternal blood and tissues.     

Ovine and Human Placental IgG inhibit Human Natural Killer Cell Cytotoxicity in vitro

To determine the effect of ovine and human placental IgG on human Natural Killer (NK) cell cytotoxicity in vitro placental IgG was eluted at acidic pH and purified by ion exchange and subsequently by affinity chromatography on protein G and protein A sepharose columns. These antibodies were analysed for presense of IgG by immuno-electrophoresis and relative purity determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS PAGE). The effect of these antibodies on human NK cell cytotoxicity was investigated by slChromium Release Assay using human K562 cells as targets and human peripheral blood lymphocytes as effector cells. Both ovine and human placental IgG inhibited human NK cell cytotoxicity in a dose dependent manner. Placental IgG may down-regulate the cytotoxic effects of NK cells in vivo by competitively excluding the binding of NK cells to their respective targets on the trophoblast. Alternatively, these antibodies may themselves be toxic to NK cells. Either way, the presence of these antibodies on the placental trophoblast may prevent the binding of NK cells and subsequent immunological rejection of the fetal allograft. Also, ovine placental IgG may be functionally similar to its human counterpart and may therefore be suitable as a model for the study of maternal fetal interaction during pregnancy in humans.     

Natural Killer Cell Cytotoxicity and Paternal Lymphocyte Immunization in Women with Recurrent Spontaneous Abortions

PROBLEM: Natural killer (NK)-cell cytotoxicity in women undergoing lymphocyte immunization prior to and following treatment was investigated. METHOD OF STUDY: A cohort of 33 women with a history of two or more recurrent spontaneous abortions was prospectively studied. NK-cell cytotoxicity was determined at effector-to-target ratios of 50:1 and 25:1. Peripheral blood CD56+ NK-cell, CD 19+ B-cell, CD19+/5+ B-l-cell, and CD3+ pan T-cell levels were studied by flow cytometry before and after lymphocyte immunization treatment. Maternal antipaternal T- and B-cell antibody levels were measured before and after lymphocyte immunization by flow cytometric analysis. Paternal lymphocyte immunizations were given two times with a 4-week interval. Post-lymphocyte immunization testing was done 4 weeks after the second lymphocyte immunization. The controls were 8 normal healthy women. NK assays were done twice with an interval of 8 weeks. RESULTS: NK-cell activity at effector-to-target ratios of 50:1 (P = 0.005) and 25:1 (P = 0.001) were significantly suppressed after lymphocyte immunization. CD3+ pan T-cell levels after lymphocyte immunization were significantly increased compared with levels before lymphocyte immunization (P = 0.008). CD56+ NK-cell levels were significantly suppressed after lymphocyte immunization (P = 0.016). There was no correlation between changes in NK cytotoxicity and differences in antipaternal lymphocyte antibody levels before or after lymphocyte immunization. CONCLUSION: Lymphocyte immunization suppresses NK-cell cytotoxicity and CD56+ NK-cell levels and increases the peripheral blood CD3+ T-cell population in women with recurrent spontaneous abortions.     

The molecular basis of Natural Killer (NK) cell recognition and function

Natural Killer cells are likely to play an important role in the host defenses because they kill virally infected or tumor cells but spare normal self-cells. The molecular mechanism that explains why NK cells do not kill indiscriminately has recently been elucidated. It is due to several specialized receptors that recognize major histocompatibility complex (MHC) class I molecules expressed on normal cells. The lack of expression of one or more HLA class I alleles leads to NK-mediated target cell lysis. Different types of receptors specific for groups of HLA-C, HLA-B, and, very recently, HLA-A alleles have been identified. While in most instances, they function as inhibitory receptors, an activatory form of the HLA-C-specific receptors has been identified in some donors. Molecular cloning of HLA-C-, HLA-B- or HLA-A-specific receptors has revealed new members of the immunoglobulin superfamily with two or three Ig-like domains, respectively, in their extracellular portion. While the inhibitory form is characterized by a long cytoplasmic tail associated with a non-polar transmembrane portion, the activatory one has a short tail asociated with a Lys-containing transmembrane portion. Thus, these human NK receptors are different from the murine Ly49, that is a type II transmembrane protein characterized by a C-type lectin domain. A subset of activated T lymphocytes expresses NK-type class I-specific receptors. These receptors exert an inhibiting activity on T cell receptor-mediated functions and may provide an important mechanism of downregulation of T cell responses.     

Inhibition of Human Natural Killer Cell and Lymphokine-Activated Killer Cell Cytotoxicity and Differentiation by Vitamin D3

Recent data suggest that vitamin D3 may be capable of immunoregulation after it is converted to an active metabolite, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). The effect of vitamin D3 and 1,25(OH)2D3 on human natural killer (NK) cells and their activation by interferon (IFN) and interleukin 2 (IL-2) was investigated. Vitamin D3 and 1,25(OH)2D3 inhibited NK cytotoxicity in a dose-dependent manner. Pretreatment of non-adherent (NA) cells at 37 degrees C for 18 h with the vitamins also led to inhibition of NK activity. Both the inhibition of NK lysis and pretreatment of NA cells were dependent on the concentrations of fetal calf serum (FCS) in the medium. The inhibition of NK activity was less effective in the presence of 10% FCS than with 1% FCS. Vitamin D3 inhibited both IFN and IL-2 activation of NK activity. However, increasing doses of IL-2 were able to abrogate the inhibition caused by vitamin D3. Vitamin D3 was able to inhibit NK activity of phytohaemagglutinin and IL-2-activated cells, and also inhibit the proliferation and lymphokine-activated killer activity induced by IL-2. NA cells pretreated with vitamin D3 did not respond well to IL-2. NA cells pretreated with low doses of IL-2 were sensitive to inhibition by vitamin D3 while those pretreated with high doses of IL-2 were not. The data presented suggest that vitamin D3 and 1,25(OH)2D3 inhibit NK activity and LAK cellular differentiation.     

Selective Inhibitory Effects of Stress Hormones on Natural Killer (NK) Cell Activity of Lymphocytes from AIDS Patients

To examine the potential role of stress hormones in the progression of HIV infections, we developed an in vitro model system that investigates the effects of cortisol, adreno-corticotropin-releasing hormone (ACTH) and β-endorphin on the natural killer cell activity of lymphocytes from normal subjects and AIDS patients. The system employs a 4 hr⁵¹Cr release assay and K562 target cells. Direct addition of cortisol (0.05, 0.1, and 0.2 μg/ml) or ACTH (10^-6 to 10^-8 M) to the mixture of effector and prelabeled target cells did not produce any significant immunoregulatory effects on the NK cell activity of normal lymphocytes. Direct addition of β-endorphin (10^-13 to 10^-17 M) to the mixture of effector and prelabeled target cells did not produce any significant immunoregulatory effects on the NK cell activity of lymphocytes from normal or AIDS subjects. However, cortisol and ACTH significantly inhibited the NK activity of lymphocytes from AIDS patients. The selective inhibitory effects of cortisol and ACTH in patients with HIV infections are consistent with a model which proposes that stress related neurohormones and/or neuropeptides may be involved in the progression of HIV infections.     

Inhibition of Natural Killer Cell Cytotoxicity by a Monoclonal Antibody Directed against Adhesion-Mediating Protein gp 90 (CD18)

Contact is required between the effector natural killer (NK) or cytotoxic killer T cell and its corresponding target in order for efficient lysis to occur. Several surface molecules of different types are involved in this process. Here we could show that Fab fragments from a murine monoclonal antibody reacting with gp 90, the human leucocyte common antigen CD18, are extremely efficient in blocking human NK of killer T cells, regardless of whether the target has or does not have the antigen. In contrast, no impact of the antibody was observed when the effector cells were of murine origin, again regardless of whether the target cell did or did not display the leucocyte common antigen. The inhibition could be shown to occur at the level of blockage of target-conjugate formation. This means that the functional display of effector/target gp 90 on the effector but not the target cell is necessary for efficient lysis to occur both in NK and killer T cell systems.     

Production and Characterization of a Novel Monoclonal Antibody Inhibitory for Murine Natural Killer Cell Activity

Natural killer (NK) cells are considered to play an important role in tumor surveillance. The killing of tumor target cells by NK cells is the result of a complex series of sequential binding, signal processing and lytic events. However, the mechanism which NK cells use to recognize tumor targets is poorly understood. To further study the cell-surface molecules involved in tumor recognition, we immunized rats against cloned murine T cells with NK activity (DBA/2.1) and generated rat-mouse hybridomas which were screened for the ability to block lytic activity of DBA/2.1 effector cells. Culture supernatants from one IgM-producing hybridoma, designated S1C4, were found to consistently inhibit DBA/2.1-mediated lysis of YAC-1 target cells. Endogenous splenic NK activity was also diminished in the presence of S1C4 monoclonal antibody (mAb) while alloantigen-specific cytotoxic T lymphocyte (CTL) activity was not affected. S1C4 mAb appears to react with effector cell-surface structures involved in the recognition/adhesion phase of NK activity since pretreatment of effector cells with mAb S1C4 inhibits their ability to bind to YAC-1 target cells. ELISA studies revealed that the S1C4 antigen is expressed by a range of lymphoid cell lines, as well as by DBA/2.1 cells and fresh splenic NK cells. S1C4 mAb were shown to react with 22, 24, 30, and 46 kiloDalton (kDa) DBA/2.1 cell membrane components on immunoblots performed under reducing conditions. These structures do not correspond to any known recognition/adhesion molecules, suggesting that mAb S1C4 defines novel cell membrane components involved in NK cell function.     

Effects of X-RAY Irradiation on Natural Killer (NK) Cell System. L Elevation of Sensitivity of Tumor Cells and Lytic Function of NK Cells

Abstract

The in vitro effect of X-ray irradiation on the human natural killer (NK) system was studied. When K562 cells were irradiated with X-rays and cultured for 18 hours, they showed increased sensitivity to lysis by blood lymphocytes and purified large granular lymphocytes (LGL). The X-ray-induced augmentation was observed as little as 2 Gy irradiation, reaching maximum at 5 to 20 Gy. The doses of X-rays did not influence the viability and spontaneous release of the target cells. On the other hand, irradiation with X-rays of NK cells at 5 to 15 Gy resulted in a transient increase in NK activity at 1 hour, and then the activity declined and was completely lost after 24 hours. However, when LGL were cultured with interferon immediately after irradiation, they maintained elevated NK activity. These results suggest the possible use of low doses of X-ray irradiation in combination with biological response modifiers for treatment of cancer.     

Effects of X-RAY Irradiation on Natural Killer (NK) Cell System. L Elevation of Sensitivity of Tumor Cells and Lytic Function of NK Cells

The in vitro effect of X-ray irradiation on the human natural killer (NK) system was studied. When K562 cells were irradiated with X-rays and cultured for 18 hours, they showed increased sensitivity to lysis by blood lymphocytes and purified large granular lymphocytes (LGL). The X-ray-induced augmentation was observed as little as 2 Gy irradiation, reaching maximum at 5 to 20 Gy. The doses of X-rays did not influence the viability and spontaneous release of the target cells. On the other hand, irradiation with X-rays of NK cells at 5 to 15 Gy resulted in a transient increase in NK activity at 1 hour, and then the activity declined and was completely lost after 24 hours. However, when LGL were cultured with interferon immediately after irradiation, they maintained elevated NK activity. These results suggest the possible use of low doses of X-ray irradiation in combination with biological response modifiers for treatment of cancer.