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Positive self regulation of cytotoxicity in human natural killer cells by production of interferon upon exposure to influenza and herpes viruses.

Augmentation of natural killer (NK) activity by influenza A/PC and HSV- 1 viruses appears to be caused by the induction of interferon (IFN) within the NK cell population itself. These viruses induced high levels of IFN production by human large granular lymphocytes (LGL) that could be readily isolated from peripheral blood by Percoll density gradients. These LGL, which have been previously shown to account for and to be highly associated with endogenous NK activity, became augmented in their lytic function during the 18-h period that IFN was induced. Non- LGL helper cells did not appear to be required in the NK-IFN system (either T cells, B cells, or monocytes). Removal of these latter cell types by treatment with OKT3 plus complement, anti-IgM plus complement, or preincubation with silica or carrageenan had no effect on the ability of LGL to respond to the viruses. Production of IFN was also detected, albeit at lower levels, from monocytes cultured for 18 h with viruses, but no cytotoxic activity was induced. On the other hand, T cells, even in the presence of monocytes, showed neither property, and longer cultures, with virus up to 4 d, still did not alter the pattern. The IFN produced by both LGL and monocytes were predominantly IFN- alpha, as assessed by neutralization assays with antisera to IFN-alpha, -beta, and -gamma. In an individual with detectable serum antibodies to influenza A/PC, however, the IFN induced in LGL appeared to be gamma, presumably because of specific recognition of the virus. These data suggest an efficient positive self-regulatory mechanism in NK cells that may be readily switched on by viruses.

subsequently become augmented in their lytic function. We also considered the ability of other cell types to provide helper function, either by IFN production themselves or by proper presentation of the virus to the IFN-producing cell, thus aiding in the augmentation sequence. This aspect was worth investigating because of previous reports that influenza virus can induce IFN in monocytes (19) and that Sendai virus might induce IFN in B cells (20), whereas HSV-1 has been shown to induce IFN in Fc receptor-negative null cells (21). We demonstrate that large granular lymphocytes (LGL), which possess virtually all of the NK activity and comprise ~ 10% of the total PBL (22), can produce IFN of either e~ or y type in response to influenza virus, and the type induced may depend on the donor's previous encounter with the virus. Herpes simplex, on the other hand, induced mainly IFN-a production by LGL. In parallel with IFN production, enhanced NK activity was seen in LGL in all cases after incubation with the viruses, suggesting positive self regulation of function.

Materials and Methods
Viruses. Influenza A/Port Chalmers/1/73 (H3N2) virus, originally obtained from Dr. Geoffrey Schild, National Institute for Biological Standards and Control, Hampstead, England, was prepared by inoculation in fertile hens' eggs (23). Herpes simplex 1 (HSV-1), Rodanus strain, was prepared by inoculation into Vero cell cultures (24). Both viruses were kindly provided by Martha Wells, Food and Drug Administration, Bureau of Biologics, Bethesda, MD.
Peripheral Blood Lymphocytes. Lymphocytes were obtained from three sources, all located at the National Institutes of Health, Bethesda, MD. The lymphocyte concentrates from the Platelet-Pheresis Center were diluted 1:5 in RPMI 1640 medium containing 10% fetal bovine serum (FBS), 100/xg/ml each of penicillin and streptomycin, 2 mM glutamine, 5 mM Hepes, and 2 U/ml of preservative-free heparin. The buffy coats from the blood bank and the leukopacks from the leukopheresis center were diluted 1:2 in phosphate-buffered saline (PBS). PBL were then prepared from the three types of blood preparations by centrifugation on Ficoll-Hypaque at 400 g for 30 min at room temperature.
Discontinuous Percoll Density Gradient Centrifugation. The separation of LGL, which are large lymphocytes with kidney-shaped nuclei and azurophilic cytoplasmic granules from small mature T cells, was accomplished by the use of a discontinuous Percoll density gradient (18). An essential step before the gradient centrifugation was the rigorous removal of adherent cells that tended to band in the lighter fractions close to those of LGL. More than 99% removal of adherent cells was achieved by incubation of the PBL obtained after Ficoll-Hypaque centrifugation on plastic petri dishes (3003; Falcon Labware, Div. of Becton, Dickinson & Co., Oxnard, CA) or tissue culture flasks (Costar Data Packaging, Cambridge, MA 3275) for 2 h at 37°C and a further incubation on nylon columns for 30 min at 37°C. The cells passing through the nylon columns were then placed on a seven-step discontinuous Percoll density gradient that varied in 2.5% concentrations from 40-55%, as has previously been described in detail elsewhere (25). Constant osmolarity of the Percoll solution and the dilution medium (285 mosmol/kg H20) was critical for the successful separation of LGL from the rest of the cells. After centrifugation at 550 g for 30 min at room temperature, the bands of lymphocytes were collected and checked for LGL morphology on Giemsa-stained cytocentrifuged slides. In this series of experiments, fraction 3 contained 70-90% LGL, and fractions 6-7 contained 295% OKT3 + T cells. Monocytes/macrophages were recovered by gentle scraping of the petri dish-adherent cells with a rubber policeman.
Culture Conditions for the Induction of lFN and Augmentation of NK Activity. Based on preliminary experiments that determined the optimum concentrations of viruses and cells, the following procedure was adopted for routine use. The cultures were carried out in RPMI 1640 medium containing 1% FBS, 100 #g/ml penicillin and streptomycin 2 mM L-glutamine, and 5 mM Hepes. 2 million cells from each of the Percoll fractions as well as an aliquot taken from the nonadherent input cell population before Percoll gradient centrifugation were incubated with medium alone or with a virus, in a final volume of 1.0 ml in 24-well plates (Costar 3524; Data Packaging) for 18 h at 37°C. The final concentrations of influenza A/PC and HSV-1 were 1:1,000 and 1:100 of the stock solutions, respectively. In addition to these cell cultures, an aliquot of each of the subpopulations was placed in medium and kept in the refrigerator for the same length of time, to provide baseline activities. In most experiments, this 4°C control did not differ from the medium control at 37°C and, therefore, those results are not reported.
At the end of the incubation, the supernatants from all the cultures were collected and frozen at -70°C or immediately tested for the content of IFN. Additional controls were included in preliminary experiments, in which the viruses were incubated in the absence of lymphocytes, and the supernatants were collected for the detection of IFN. These controls were necessary because of the possibility that the viruses might induce IFN production in the human fibroblast test cells during the IFN titrations, thus, giving false positive results. However, the virus supernatants were consistently negative in the IFN assay. After collection of the supernatants, the cells were harvested and transferred to 96-well microtiter plates for mesasurement of NK activity.
Removal of T Cells. To eliminate contaminating T cells from the LGL fractions, 0.2 ml of a 1:4 dilution of OKT3 (Ortho Pharmaceutical, Raritan, N J) was added to 1 × 10 7 lymphocytes in 0.1 ml of medium. The mixture was kept on ice for 30 min before addition of 0.1 ml of a 1:4 dilution of rabbit complement (Gibco Laboratories, Grand Island Biological Co., Grand Island, NY). After further incubation for 45 min at 37°C in a shaking water bath, the cells were washed and resuspended to 4 × 106 cells/ml. No correction of the viability was done, so that the original number of cells that could produce IFN or be boosted could be retained. Medium and complement controls from the same fractions were included in most of the experiments. In addition, to check for the efficiency of the procedure for removal of T cells, an aliquot of the nonadherent input cells that contained a large proportion of T cells as well as an aliquot of T cells from fraction 7 were similarly treated, with the appropriate medium and complement controls. Cell viability was then checked on all samples.
Removal of B Cells. The procedure was identical to that mentioned above for OKT3, except that that the antibody used was goat anti-IgM (N. L. Cappel Laboratories, Cochranville, PA), and 0.1 ml of the undiluted reconstituted serum was added to 1 × 10 7 lymphocytes (26). Because nylon wool-passed nonadherent cells were virtually free of B cells, additional controls consisting of whole unseparated PBL were included to check for the proper elimination of B cells. Cell viability was determined in all samples after the various treatments.

Inactivation of Monocytes.
Contaminating monocytes in the LGL preparations were inactivated by incubation with silica or carrageenan at final concentrations of 200/lg/ml, 100/~g/ml or 50 ~g/ml for 2 h at 37°C (27).
Titration of IFN. The levels of IFN in the culture supernatants were determined by the inhibition of cytopathic effect by vesicular stomatitis virus in human foreskin fibroblasts when performed at the Bureau of Biologics (28) and by Sindbis virus in human WISH amniotic cells when performed at the University of Texas (26). The titers are expressed in international units, standardized with NIH reference human leukocyte IFN (G-023-901-527).
Qualitative identification of IFN in the culture supernatants was performed by incubating each IFN sample with 10-100-fold excess of antiserum of each type for 1 h at room temperature. In some experiments, two or more antisera were added to determine the presence of a mixture of IFN species. The IFN/anti-IFN mixture was then diluted serially at half-log dilutions through six wells of a 96-well microtiter plate. These dilutions were then transferred to wells containing human fibroblasts or WISH cells for determination of residual IFN activity. Laboratory standards of IFN-a, fl, y were included in each assay.
Measurement of NK Activity. NK activity was determined by the release of radioactivity from 5 × 103 5ICr labeled K562 tumor cells after 4 h incubation at 37°C, with lymphocytes at effector/target ratios of 50:1 and 10:1 in a 96-well microtiter plate (25). The supernatants were collected by the Titertek harvesting system (Flow Laboratories, McLean, VA). The baseline (autologous) lysis was determined by the addition of unlabeled autologous tumor cells to the labeled targets at the same ratios as lymphocytes to target cells. Percent NK activity was then calculated as follows: ([experimental epm -autologous cpm]/[total cpm incorporation]) × 100.
The autologous control was usually 6-8%, and the standard error in most groups was <1%. Percent cytotoxicity of >2.0% was usually significantly different from the control at P < 0.05. For clarity in presenting data in Results, the standard errors were not included, and percent eytotoxicity <1% was expressed as 0% in all cases.

Induction of IFN and Boosting of NK Activity in Cell Fractions Recovered from Percoll
Gradients. Results of initial studies to define the cell population(s) that responded to influenza A/PC and HSV-1 are shown in Fig. 1. The fractions incubated with medium alone showed the typical profile of distribution of spontaneous NK activity, with peak activities in fractions 2-3, in which 70-90% of the cells showed LGL morphology on cytocentrifuged slides. The denser cells, particularly in fractions 5-7, which consisted mainly of small lymphocytes with the characteristics of mature T cells, had no spontaneous NK activity. In the presence of the two viruses, the

100
,,=, FIG. 1. Augmentation of NK activity and induction of IFN in lymphocytes fractionated by discontinuous Percoll gradients. Each lymphocyte fraction was incubated with medium (O), influenza A/PC virus (i), or HSV-1 ((3) for 18 h at 37°C before they were tested for production of IFN and for augmentation of NK activity at a 10/1 effector/target ratio. nonadherent input cells showed IFN production and boosted NK activity. After these cells were fractionated according to density, only the LGL-enriched fractions 2-3, which initially contained spontaneous NK activity, responded by IFN production. The level of IFN detected was remarkably high, particularly in LGL cultures containing influenza A/PC. As expected, NK activity was also augmented in these same fractions. There was no quantitative correlation between the level of IFN production and the degree of augmentation of NK activity, suggesting (as has been previously reported) (5) that small amounts of IFN may suffice to produce maximum cytotoxicity. IFN was also detected in fraction 1, which was probably produced by monocytes, as will be seen in more detail in experiments to be discussed below. A consistent observation was the lack of IFN production or boosted cytotoxicity in T cells of fractions 5-7.
Kinetics of IFN Production and Boosting of NK Activity. The lack of a T cell response might have been due to insufficient time of incubation with the viruses. Moreover, proper presentation of the virus by adherent accessory cells might have been necessary for stimulation of T cells. To rule out these possibilities, LGL from fraction 3 and T cells from fractions 6-7 were cultured in the absence or presence of 10% monocytes for 1-4 d, with optimum doses of influenza A/PC or HSV-1. In parallel, adherent monocytes, either at the 10% level or at a 10-fold higher concentration, were incubated with viruses (Table I). Again, the LGL responded to both viruses by IFN production and augmented cytotoxicity, and peak levels were observed by day I. Addition of monocytes did not enhance either function, although monocytes were also capable of producing IFN. It is interesting to note that monocytes, at the concentration added to provide helper function or at the 10-fold higher concentration, still did not produce the levels of IFN production seen with LGL. T cells showed no response in the presence of monocytes, even after 4 d of culture. The low levels of IFN seen in the mixture of T cells and monocytes can be accounted for by production by the monocytes alone.

Elimination of Contaminating T Cells from
LGL. Although small mature T cells were not involved in IFN production under the conditions of our assay, other T cells of  medium size that could contaminate the LGL fractions might serve as the source. To eliminate this possibility, LGL and the nonadherent input ceils were both treated with OKT3 plus complement before incubation with influenza A/PC or HSV-1. As seen in Table II, this treatment eliminated 75% of the input cells, 100% of the cells in fraction 7, and 25% in the LGL-containing fraction. (In other experiments, 10-40% of the LGL fractions were killed, depending on the purity of the samples. This level of OKT3-positive cells is often seen in LGL fractions that have not been further fractionated with 29 ° E rosetting). The treated LGL were still as potent as the medium or complement control cells in producing IFN and exhibiting enhanced cytotoxicity. The input fraction, when treated with OKT3 plus C, often produced low levels of IFN, (~25 U) in the absence of either virus. This low level might be due to the IFN-inducing capability of OKT3 itself on T cells. An accompanying enhancement of NK activity was also seen.

Elimination of Contaminating Monocytes from
LGL. Because LGL-enriched fractions contain small percentages of monocytes, it was necessary to further eliminate the possibility that some monocytes contaminating the LGL fractions might have provided helper function or served as the IFN source. Therefore, the LGL-enriched fractions were incubated with silica or carrageenan for 2 h at 37°C and then cultured with influenza A/PC or HSV-1. Monocytes alone were also treated in a similar fashion, to monitor the efficacy of the monocyte-toxic agents. As seen in Table III, monocytes produced 125 U and 50 U/ml of IFN with influenza A/PC and HSV-1, but this function was lost after treatment with 100-200/~g/ml of silica or 200 #g/ml of carrageenan. Of interest was the ability of silica to inhibit NK activity in LGL but not IFN production, when used at 200 #g/ml. Silica at lower doses and carrageenan at all doses used did not interfere with either of the functions of LGL. Further proof of the ability of LGL alone to respond to viruses was provided by the simultaneous treatment of LGL with OKT3 plus complement and silica (Table IV). Removal of both T ceils and monocytes from LGL had no effect on the IFN-producing capability or the enhancement of cytotoxicity.

Elimination ofContarninating B CeUsfrorn
LGL. The input cells used in the fractionation procedure had first been passed through nylon wool, which is highly efficient in removing B cells. Because there have been some reports that B cells are capable of producing IFN, particularly to Sendai virus (20) or to heterologous tumor cells (26), we examined the possibility of a small amount of contamination of LGL with B ceils

Monocytes
LGL #g/ml that might contribute to IFN production.
LGL were thus treated with anti-IgM plus complement, before incubation with the viruses. Table V shows that ~20% of PBL but <1% of nylon wool-passed cells and <1% of LGL were killed by this treatment.
The treated cells showed unimpaired IFN production and enhanced NK activity.
Characterization of IFN. Antigenic typing of the IFN produced by LGL and monocytes in response in influenza A/PC and HSV-1 was performed in two separate laboratories, at the University of Texas (Table VI) and at the Bureau of Biologics (Table VII), using different sources of antisera prepared against IFN a, fl, and 7. The data from both laboratories were comparable and showed that the IFN produced in LGL or monocytes in most cases was readily neutralized by anti-a and not by anti-fl   Table VII. Further analysis of one of these individuals showed that he was seropositive to influenza A/PC.

Discussion
Recently available techniques using discontinuous Percoll density centrifugation for isolation of human LGL, which are primarily responsible for NK function in PBL, have enabled us to examine the NK-IFN system in detail. In particular, we have been able to direct our research to answer the questions: * Cell culture supernatants diluted to an initial concentration of 125-250 IFN U/ml were preincubated with each antiserum for 1 h at room temperature before they were titered for IFN on human foreskin fibroblasts challenged with vesicular stomatitis virus. :~ Anti-a serum was prepared by K. Zoon and colleagues. § Anti-fl serum was obtained from Dr. Y. K. Yip and Dr. J. Vilcek. II Anti-3' serum was prepared by G. J. Stanton and colleagues. acquire NK function; and (e) whether more than one species of IFN can be induced in a single subset of lymphoid cells. The results presented here showed that LGL were remarkably well-equipped to respond to influenza and HSV-1. The response was twofold: a rapid induction of IFN and a parallel enhancement of NK activity. Peak levels of IFN, ~ 1 × 10 a U/106 cells, were induced within 24 h of exposure to either virus, and addition of monocytes to the cultures did not enhance IFN production or raise NK levels further. Other lymphocyte fractions isolated from the Percoll gradient, which made up 90% of the input nonadherent cells, exhibited neither IFN nor NK function in the absence or presence of viruses. We considered the possibility that the failure to observe some response in these non-LGL fractions might have been due to either of two factors (a) a slower kinetics in the production of IFN; or (b) the lack of a helper population. However, incubation for up to 4 d, with or without the addition of monocytes, did not alter the pattern, and no IFN or lyric activity was observed in the small T cell fractions.

(a) whether IFN may be produced by LGL in response to inducers such as viruses; (b) whether helper cells may be required for optimum IFN production in LGL; (c) whether other classes of lymphoid cells may produce virally-induced IFN; (d) whether IFN can activate non-LGL to
On the other hand, monocytes responded to the viruses by IFN production, but they did not acquire the ability to lyse K562 target cells. The levels of IFN were usually below those produced in LGL. The ability of monocytes to produce IFN during overnight culture with influenza virus has been described earlier (19), and our data are consistent with that previous report. It is interesting to note the IFN produced in leukocytes on a large scale for clinical trials are virally induced and might in large part be a product of LGL, with only a smaller contribution from monocytes.
The purity of the LGL population was critical for definitive identification of these ceils as the source of viral-induced IFN. To leave little doubt about the capability of LGL to produce IFN, the LGL fractions were subjected to further purification by removal of contaminating T cells, monocytes, and B cells. Removal of T cells from the LGL by monoclonal OKT-3 plus complement had no effect on IFN production or enhancement of NK activity. Similarly, incubation of LGL with 100/.tg/ml of silica or various concentrations of carrageenan, which inactivated monocytes, did not interfere with the two functions. Of interest was that higher levels of silica could inhibit spontaneous NK activity in LGL, but their IFN-producing capability was not blocked. This result might explain previous in vivo observations of potent inhibition by silica of NK activity in mice (32). It might represent a direct sublethal toxic effect of silica on NK cells rather than the elimination of monocytes that are required for the maintenance of in vivo NK activity. Simultaneous treatment of LGL with OKT3 plus complement and silica provided further proof of the ability of LGL to function alone in response to viruses.
Analysis of the antigenic characteristics of the IFN induced by the viruses with a battery of antisera against IFN a, fl, and 3, showed that LGL and monocytes, in most individuals, produced IFN-a to both viruses. However, the LGL of a individual with detectable serum antibodies against influenza A/PC, indicating previous exposure to the virus, produced IFN-y. This is consistent with the recent report of Ennis et al. (33), who used unfractionated PBL from volunteers who were vaccinated with influenza. Our results are also in line with those of Greene et al. (34), who observed only IFN-a in HSV-seronegative individuals but IFNa and IFNy in seropositive individuals when their PBL were stimulated with HSV-1. A surprising finding was the high levels of IFN-a, and particularly of IFN-T, from LGL cultured with influenza A/PC. We routinely detected 1,000 U/106 LGL and at times obtained 10,000-15,000 U/10 n LGL, similar to the findings of Ennis et al. (33), suggesting that this might be an efficient means of producing IFN-), in quantities large enough for research or clinical purposes. The other quite intriguing aspect of this suggestion of IFN-), production by LGL is that it provides an indication for a specific-immune function by these cells, which have been considered by us and some other investigators to be within the T cell lineage.
The observation that LGL responded to influenza and HSV-1 by IFN production is an important finding because of the implication that NK cells are under positive self-regulation, being able to produce IFN that can in turn activate cytolytic function of these cells. The efficiency of the NK cell system is reflected in their apparent lack of a requirement for helper cells of another cell type, either for the IFN production stage or for the activation of the lytic mechanism. A second finding of equal importance was the lack of augmentation of cytotoxicity in T cells, even in the presence of monocytes that can provide IFN as the boosting agent. IFN appears to enhance cytolytic function only in LGL, indicating that IFN-activatable precursor NK cells do not exist in non-LGL fractions. There has been some suggestive evidence that the human cell type that produced IFN on exposure to virus-induced tumor cells was the same as the cytotoxic effector cell (12). Our results now firmly establish human LGL as the source of both virus-induced IFN production as well as NK activity. These findings mirror observations made in the mouse that IFN and NK activity are confined to the same set of Ly5 +, Qa5 +, asialo GM1 + spleen cells (17). However, it remains to be determined whether the same subpopulations of LGL with NK activity also are responsible for the IFN production in response to viruses.
Our data do not indicate whether there is a recruitment of new NK cells from an inactive population in the LGL fraction or whether cytolytic activity is enhanced in already active NK cells. Minato et al., in the mouse, have shown a conversion of precursor NK to the active state (17). In the human, Targan and Dorey (35) as well as Timonen et al. (36) have shown that both recruitment of new effector cells and activation of lytic function take place. Timonen et al. (36), in particular, have shown in great detail that the two phenomena occur in the LGL population. Future experiments with cloned LGL cultures should provide a better insight into the exact mechanism of NK augmentation.
Another aspect of considerable interest in the present findings was the ability of LGL to produce more than one type of IFN. It would be of interest to determine whether the different types of IFN are made by separate subpopulations or whether, depending on the nature of and previous exposure to the stimulus, the same cells can produce different IFN species. In any event, these data strengthen the suggestion that NK cells might be important in natural resistance to various microorganisms, particularly in the early stage of infection.

Summary
Augmentation of natural killer (NK) activity by influenza A/PC and HSV-1 viruses appears to be caused by the induction of interferon (IFN) within the NK cell population itself. These viruses induced high levels of IFN production by human large granular lymphocytes (LGL) that could be readily isolated from peripheral blood by Percoll density gradients. These LGL, which have been previously shown to account for and to be highly associated with endogenous NK activity, became augmented in their lytic function during the 18-h period that IFN was induced. Non-LGL helper cells did not appear to be required in the NK-IFN system (either T cells, B cells, or monocytes). Removal of these latter cell types by treatment with OKT3 plus complement, anti-IgM plus complement, or preincubation with silica or carrageenan had no effect on the ability of LGL to respond to the viruses. Production of IFN was also detected, albeit at lower levels, from monocytes cultured for 18 h with viruses, but no cytotoxic activity was induced. On the other hand, T cells, even in the presence of monocytes, showed neither property, and longer cultures, with virus up to 4 d, still did not alter the pattern. The IFN produced by both LGL and monocytes were predominantly IFN-a, as assessed by neutralization assays with antisera to IFN-cL -iS, and -y. In an individual with detectable serum antibodies to influenza A/PC, however, the IFN induced in LGL appeared to be T, presumably because of specific recognition of the virus. These data suggest an efficient positive self-regulatory mechanism in NK cells that may be readily switched on by viruses.