David Coggins, Frederick Cancer Research Center, Freder- ick, Md., and represent the number of cells per 107 effectors required to achieve 20% lysis of the ...
INFECTION AND IMMUNITY, Mar. 1992, p. 853-863
Vol. 60, No. 3
0019-9567/92/030853-11$02.00/0 Copyright ©) 1992, American Society for Microbiology
Growth Inhibition of Candida albicans by Interleukin-2-Activated Splenocytes D. W. A. BENO AND H. L. MATHEWS*
Department of Microbiology and Immunology, Stritch School of Medicine, Loyola University of Chicago, Maywood, Illinois 60153 Received 25 September 1991/Accepted 26 December 1991
Murine splenocytes, Percoll-enriched low-density lymphocytes, and interleukin-2 (IL-2)-activated lymphowere assessed for the capacity to limit the growth of the hyphal form of Candida albicans. No fungal-growth-inhibitory activity was exhibited for C. albicans by either splenocytes or Percoll-enriched lymphocytes. These cells were capable of cytotoxic activity for a natural killer cell-sensitive cell line. However, when cultured for several days with IL-2, splenocytes acquired the capacity to inhibit the growth of the fungus. The appearance of the antifungal activity coincided with the development of cytotoxic activity for the natural killer cell-insensitive cell line. Anti-C. albicans and antitumor activities of IL-2-activated lymphocytes were competitively and reciprocally inhibited by C. albicans and the natural killer cell-sensitive and -insensitive cell lines. The antifungal activity of the IL-2-activated lymphocytes was exhibited against a number of clinical isolates of C. albicans and related fungal species. IL-2-activated human peripheral blood lymphocytes also acquired the capacity to inhibit the growth of C. albicans. These data show that in vitro growth inhibition can be mediated by IL-2-stimulated lymphocytes which are neither fungal strain nor mammalian species restricted in their biological activity. cytes
splenic clearance of the fungus (2). A correlation does exist between cytokine production and mouse strain resistance to C. albicans. Neta and Salvin have shown the release of cytokines in mice sensitized with cell walls of C. albicans to be genotypically related to inbred mouse strain type (30). They found that the release of cytokines parallels the capacity of a particular mouse strain to resist intravenous infection with C. albicans. C. albicans-infected strains of mice that released high levels of interleukin-2 (IL-2) and gamma interferon (highly resistant strains such as C57BL/6) were shown to have lower numbers of C. albicans in tissue than did those strains of mice which released lower levels of these cytokines (30). The physiological role of cytokine-activated lymphocytes in C. albicans infection is unclear. Cytokine-activated lymphocytes have been shown to be present in fluids isolated from inflammatory sites (13) and at low frequency in the peripheral blood of healthy individuals (20). These cells may play a role in host resistance to infectious disease. Cytokineactivated lymphocytes can be isolated from mice inoculated with either C. albicans or its cell wall constituents (3). These lymphocytes possess unrestricted cytotoxic activity for a variety of tumor cell lines. However, those lymphocytes have not been evaluated for anti-C. albicans activity. In this investigation we sought to identify culture conditions for optimal development of antifungal activity and cell recovery of murine lymphocytes which mediate growth inhibition of C. albicans. Once these conditions were identified, we sought to determine the following: whether the optimal antifungal activity correlated biologically with natural killer cell (NK) activity, whether the antifungal activity could be competed for by NK-resistant and NK-sensitive cell lines, whether the antifungal activity was C. albicans strain dependent, and whether the antifungal activity could be induced by IL-2 activation of human peripheral blood lymphocytes. The C57BL/6 mouse strain was selected because it is highly resistant to C. albicans.
Candida albicans is an opportunistic pathogen with a wide of infectious capabilities. Infections vary from localized to systemic and are potentially fatal. The immune response to C. albicans varies directly with the type of C. albicans infection. Localized infections are controlled by polymorphonuclear leukocytes (PMNs) and later by macrophages (34, 37). Invasive infections of C. albicans are the result of either the hyphal or pseudohyphal forms of the fungus (35). These microbial forms are too large to be ingested by phagocytic cells but have been shown to be damaged by PMNs and macrophages via extracellular mechanisms (9, 10). Host defense against C. albicans is mediated by phagocytic cells as well as by nonphagocytic cell populations, and the relative contribution of each cell population in protection against specific forms of candidiasis remains to be elucidated (4, 6, 37). Multiple cell populations have been implicated in the immune response to systemic fungal infection, and evidence for the absolute involvement of any one type of effector cell population is inconclusive. Animal models with defects in lymphoid populations have failed to elucidate a precise anti-C. albicans mechanism (21). Therefore, it is possible that nonphagocytic cell populations, activated by the immune response to C. albicans, contribute to the immunologically protective response to C. albicans. The C57BL/6 mouse strain is highly resistant to infection with C. albicans. Other inbred murine strains are more susceptible to the fungus (22). The differential susceptibility between murine strains is not due solely to differences in phagocytosis or splenic clearance of the microorganism (1). Murine strains such as C57BL/6 (highly resistant to C. albicans infection), BALB/c (intermediately resistant to C. albicans infection), and CBA/H (poorly resistant to C. albicans infection) show no correlation between survival following C. albicans challenge and either phagocytosis or range
*
Corresponding author. 853
854
BENO AND MATHEWS
MATERIALS AND METHODS Mice. C57BL/6 female mice, ages 6 to 7 weeks, were obtained from Jackson Laboratory, Bar Harbor, Maine. Mice were 6 to 12 weeks of age when used in experiments. Tumor cell lines. The P815 mastocytoma cell line was obtained from the American Type Culture Collection, Rockville, Md. P815 cells used for assessment of cytotoxic activity were maintained in suspension cultures in vitro in Corning 25-cm2 tissue culture flasks (Corning Glass Works, Corning, N.Y.) in RPMI 1640 (GIBCO Laboratories, Grand Island, N.Y.) supplemented with 10% fetal bovine serum (FBS), low levels of lipopolysaccharide (GIBCO Laboratories), 100 U of penicillin per ml, 100 ,ug of streptomycin (Whittaker M. A. Bioproducts, Walkersville, Md.) per ml, 0.1 mM nonessential amino acids, and 2 mM L-glutamine (GIBCO Laboratories). This medium was used throughout, except where noted, and is referred to as culture medium. The YAC-1 cell line was obtained from J. Clancy, Loyola University Medical Center, Maywood, Ill. Cultured YAC-1 cells were maintained in a manner identical to the P815 cell line. Fungal culture. C. albicans ATCC 58716, obtained from T. Hashimoto, Loyola University Medical Center, was used throughout this investigation (15). Cultures were stored at 25°C on Sabouraud dextrose agar (Becton Dickinson and Co., Cockeysville, Md.). Cells used for experimentation were cultured overnight at 37°C on Sabouraud dextrose agar, collected as isolated colonies, and washed once in Hanks' balanced salt solution (HBSS). Yeast cultures were enumerated microscopically, and those with more than 15% budding were discarded. C. albicans cells were inoculated into RPMI 1640 prepared as described above, with 1% FBS. C. albicans hyphal forms were obtained by incubation at 37°C, with 5% CO2 in RPMI 1640. An inoculum of 105 yeast cells per ml yielded approximately 100% hyphal fragments ranging in length from 30 to 50 ,um when incubated for 2 h at 37°C. Clinical isolates of Candida spp. and related fungi were obtained from the Clinical Laboratories at the Loyola University Medical Center. IL-2 activation of lymphocytes. Spleen cells were placed in culture medium containing 5 x 10-5 M 2-mercaptoethanol (2-ME) at a concentration of 2.5 x 106 cells per ml with 1,500 U of IL-2 (Hoffmann-La Roche Inc., Nutley, N.J.) per ml in Falcon Multiwell plates (Becton Dickinson, Lincoln Park, N.J.). This concentration of IL-2 was determined to be optimal as described previously (7, 19). The cells were harvested following incubation at 37°C, overlaid onto Lymphocyte Separation Medium (Litton Bionetics, Kensington, Md.), and centrifuged at 1,000 x g for 20 min. The cells at the interface were washed twice with HBSS prior to assessment of cytocidal or growth-inhibitory activity. These lymphoid cells were >99% lymphocytes as judged by WrightGiemsa staining. Human peripheral blood mononuclear cells were obtained by venipuncture and isolated with lymphocyte separation medium (Litton Bionetics) as described above. The cells were cultured and processed identically as described above for mouse splenocytes. Kinetic analysis of cell recovery and proliferation. Freshly isolated spleen cells were placed in culture medium and harvested (as described above) sequentially from day 1 through day 9 of culture. The total number of recoverable cells divided by the original number of cells placed in culture, multiplied by 100, was the percent recovery. Lymphoid cells were plated in 96-well flat-bottom microtiter
INFECT. IMMUN.
plates (Corning Glass Works) at 5 x 105 cells per well with or without 300 U of IL-2 per well and then pulsed with 1 ,uCi of [3H]thymidine (ICN Radiochemicals, Irvine, Calif.) per well. After 6 h of incubation, the cells were harvested with a PHD cell harvester (Cambridge Technology, Cambridge, Mass.) and associated radioactivity was determined with a Beckman LS5801 liquid scintillation counter (Beckman Inc., Irvine, Calif.). The proliferation index was defined as the incorporation of [3H]thymidine in the experimental wells containing IL-2 (300 U/ml) divided by the incorporation of [3H]thymidine in the control wells without IL-2. Percoll separations. Splenocytes were fractionated with 70 to 50% Percoll gradients (Sigma Chemical Co., St. Louis, Mo.) by centrifugation at 1,500 x g for 30 min. Each cell fraction was collected, washed twice in HBSS, and resuspended in RPMI 1640 with 1% FBS prior to experimentation. Cell fractions were designated as follows: fraction 1 represented the population banding above the 50% gradient, fraction 2 represented the population banding between the 50 and 60% gradients, and fraction 3 represented the population which had penetrated below the 60% gradient. Tumor cell cytotoxicity assay. Tumor cell lines maintained in vitro were washed once in culture medium, pelleted by centrifugation at 500 x g for 10 min, and resuspended in approximately 0.1 ml of culture medium. One hundred microcuries of 51Cr (New England Nuclear, Boston, Mass.) was added to 107 cells in a final volume of 0.2 ml. The cells were incubated at 37°C with 5% CO2 for 1 h, with agitation every 10 min. The cells were washed four times in HBSS and resuspended to 5 x 105 cells per ml in culture medium; 0.01 ml (5 x 103 cells) was aliquoted to each well of a 96-well, round-bottom assay plate (Corning Glass Works). Radiolabelled YAC-1 or P815 cells were cultured for 4 h with lymphoid cells. Following 4 h of incubation, the supernatants were removed with a Skatron harvesting press (Skatron Inc., Sterling, Va.), and associated radioactivity was determined. Maximum release was obtained by adding 0.05% Nonidet P-40 (Sigma Chemical Co.). Results are expressed as percent cytotoxicity and were calculated by the following formula: % cytotoxicity = [(experimental dpm - minimum dpm)/(maximum dpm - minimum dpm)] x 100, where dpm is disintegrations per minute. All experimental means were calculated from triplicate values. Lytic units were calculated by a program written by David Coggins, Frederick Cancer Research Center, Frederick, Md., and represent the number of cells per 107 effectors required to achieve 20% lysis of the targets (33). Inhibition of C. albicans growth by the uridine method. Fungal cells used for experimentation were collected from isolated overnight colonies on Sabouraud dextrose agar and washed once in HBSS. Yeast cells were resuspended to 2 x 105/ml in RPMI 1640 with 1% FBS, and 1 x 104 yeast cells were placed in individual wells of 96-well flat-bottom plates (no. 25861; Corning Glass Works). These plates were incubated at 37°C in 5% CO2 for 2 h to obtain C. albicans hyphal forms. Murine effector cells were added at effector/target (E:T) ratios of 50:1 to 6:1 and incubated together for 3 h at 37°C in 5% CO2. At the end of this period, murine effectors were removed by washing with a PHD cell harvester (Cambridge Technology). To the hyphae were added 0.05 ml of RPMI 1640 with 1% FBS and 1 ,uCi of [3H]uridine per well. This procedure is a modification of a previously published method (38). Following 1 h of incubation at 37°C, 25 U of lyticase (Sigma Chemical Co.) in 50 RI of HBSS was added per well and the mixture was incubated for 0.5 h at 25°C. Cells were then harvested by using a PHD cell harvester,
VOL. 60, 1992
ANTIFUNGAL ACTIVITY OF IL-2-ACTIVATED SPLENOCYTES
and associated disintegrations per minute were determined. Lyticase loosens the hyphae and permits them to be aspirated onto glass fiber filter strips. Visual inspection of the 96-well plates demonstrated that all of the hyphal forms were removed from the plastic plate. Total radioactivity associated with fungal hyphae was not affected by either the lyticase treatment or aspiration with the automated cell harvester employed as described above (data not shown). No detachment of hyphae was observed prior to the addition of lyticase. Results are expressed as percent growth inhibition and were calculated by the following formula: % growth inhibition of C. albicans = {[dpm of C. albicans alone - (dpm of sample - dpm of lymphocytes alone)]/dpm of C. albicans alone} x 100, where dpm is disintegrations per minute. All cultures were prepared at least in triplicate, and the mean percents inhibition of those values were determined. Inhibitory units, a modification of the lytic unit method (33), were calculated as the number of cells per 107 effectors required to achieve 20% inhibition of the fungal target. Inhibitory units were calculated as dpm of C. albicans alone - dpm of experimental sample. Inhibitory units were utilized for the calculation of growth inhibition with a program written by David Coggins. Cold target competitive inhibition experimentation. Cold target competitive inhibition is a modification of a previously published method for investigating interactions between C. albicans and mammalian NK (40). Tumor cytotoxicity and C. albicans growth inhibition assays were accomplished at E:T ratios of 40:1. Cultured tumor targets (P815 and YAC-1 cells) and heat-inactivated (5 min at 100°C) C. albicans hyphae were utilized as cold target inhibitors. Cold target inhibitors were washed twice in HBSS prior to coincubation with murine effectors and target cell populations ranging from 1 x 104 to 5 x 106 cells. Naive thymocytes were utilized as irrelevant mammalian targets which served as nonreactive comparisons for the other potential competitors. Thymocytes were added to each well such that the final concentration of cold targets and thymocytes of each well totalled 5 x 106 cells per well. Tumor cell line culture supernatants were collected from both overnight and 3-day cultures as well as from cultures of 5 x 106 tumor cells in 0.1 ml from 96-well plates following a 4-h culture period at 37°C. Similar supernatants were obtained from IL-2-activated lymphocytes. The supernatants were assessed for inhibition of cytotoxicity and C. albicans growth inhibition. C. albicans supematants were collected following heat inactivation. These supematants were assessed for their inherent antitumor or antifungal activities in vitro as well. The purpose of supernatant assessment was to control for potential inhibitory actions of soluble factors. Statistics. Comparisons were performed by Student's t test analysis. RESULTS Antifungal and antitumor activity of splenocytes. The purpose of initial experiments was to determine the inherent antifungal activity of murine splenocytes (Fig. la). Splenocytes possessed no growth-inhibitory activity against C. albicans as judged by inhibition of [3H]uridine uptake. Nor was there activity against the NK-resistant cell line P815 as judged by 51Cr release. Low levels of activity were observed for the NK-sensitive cell line YAC-1. This activity could be enriched by discontinuous Percoll density gradient selection (Fig. lb). Splenocytes from fraction 1 (cells which did not
855
enter a 50% concentration of Percoll) showed enhanced activity against the YAC-1 tumor cell line compared with normal splenocytes (P < 0.05). No increase in anti-YAC-1 cell activity was noted for the other Percoll-separated splenocytes. No enrichment of activity was noted for any of the other Percoll-separated cell populations against either C. albicans or P815 cells. These data demonstrate no anti-C. albicans or anti-P815 cell activity for either splenocytes or Percoll-separated cell populations. The anti-YAC-1 cell activity of normal splenocytes was enhanced in the low-density Percoll fraction. Induction of antifungal and antitumor activity in splenocytes with recombinant IL-2. Previous studies have shown that activation of lymphocytes by IL-2 can augment their antitumor cytotoxic activity (14, 16). Anti-C. albicans and antitumor activities of splenocyte populations activated with IL-2 for 1, 3, or 7 days were determined. Following 1 day of culture with IL-2, no increase in either anti-C. albicans activity or anti-P815 cell activity was noted compared with activities of day 0 splenocytes or splenocytes cultured for 1 day without IL-2 (uninduced) (P > 0.05) (data not shown). However, an increase in anti-YAC-1 cell activity was observed and was significant compared with that of day 0 splenocytes or splenocytes cultured for 1 day without IL-2 (P < 0.05). Following 3 days of culture with IL-2, anti-C. albicans activity was increased significantly compared with the activity observed on day 0 or the activity of splenocytes cultured for 3 days without IL-2 (P < 0.05) (data not shown). Anti-P815 cell activity of splenocytes was also increased significantly at day 3 of culture (P < 0.05). Splenocytes cultured without IL-2 showed no anti-P815 cell activity. Anti-YAC-1 cell activity was enhanced significantly from days 0 to 3 of incubation with IL-2 (P < 0.05). No antiYAC-1 cell activity was observed for splenocytes cultured for 3 days without IL-2 (P < 0.05). Following culture of splenocytes with IL-2 for 7 days, C. albicans-growth-inhibitory activity and tumor cytotoxic activity were significantly increased compared with those of day 0 splenocytes (P < 0.05) (Fig. 2). No cells were recovered from splenocytes cultured without IL-2 for more than 3 days. The activity of splenocytes cultured for 7 days with IL-2 was increased significantly (P < 0.05) compared with that of splenocytes cultured for 3 days for all targets examined. Cell-free supernatants from lymphocytes activated with IL-2 for either 3 or 7 days at IL-2 concentrations as high as 10,000 U per well had no antifungal activity (data not shown). These data demonstrate lymphocyte-mediated antifungal activity when splenocytes are cultured with IL-2 for extended periods of time. Kinetic analysis of lymphoproliferation, cell viability, antifungal activity, and antitumor activity of splenocytes following induction with IL-2. Splenocytes were analyzed for anti-C. albicans activity and tumor cytotoxic activity during 9 consecutive days of culture with IL-2. In experiments separate from those described in the legend to Fig. 2, IL-2activated splenocytes acquired antitumor activity against both the YAC-1 and P815 cell lines, with peak activity occurring at day 7 of culture (Fig. 3a). A significant increase in anti-YAC-1 cell activity was noted as early as day 1 compared with that of day 0 splenocytes (P < 0.05). AntiP815 cell activity was not significantly increased over that of the day 0 splenocyte population until day 3 of culture, with peak activity at day 7 (P < 0.05). C. albicans-growthinhibitory activity peaked at day 7 of culture, with significant growth-inhibitory activity appearing by day 3 compared with that of day 0 splenocytes (P < 0.05). In parallel experiments,
856
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FIG. 1. (a) C. albicans growth inhibition and tumor cell cytotoxicity of naive (day 0) splenocytes and (b) C. albicans growth inhibition and tumor cell cytotoxicity of Percoll-fractionated day 0 splenocytes. Day 0 splenocytes were fractionated by discontinuous Percoll density gradient separation. Cells from each interface were harvested and washed. Fraction (FRACT.) 1 banded above a 50% gradient, fraction 2 banded above a 60% gradient, and fraction 3 penetrated a 70% gradient. UNFRACT. refers to unfractionated cells. C. albicans growth inhibition was assessed by incorporation of [3H]uridine. Data are presented as percent inhibition and inhibitory units (IU) per 107 cells at 20% inhibition. YAC-1 cell cytotoxicity is expressed as percent cytotoxicity as judged by 51Cr release and by lytic units (LU) per 107 cells at 20% cytotoxicity. P815 cell cytotoxicity is expressed as percent cytotoxicity as judged by 51Cr release and by lytic units (LU) per 107 cells at 20% cytotoxicity. The asterisk indicates significant difference (P < 0.05) between fraction 1 activity and unfractionated splenocyte activity. Nonsignificant comparisons are not labelled; all significant comparisons are labelled. Data are expressed as the means the standard deviations for three independent experiments.
VOL. 60, 1992
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maximal cell recovery coincided with maximal antifungal activity on day 7 (Fig. 3b). Maximal proliferation of splenocytes was on day 4 of IL-2 activation and 3 days prior to maximal cell recovery. Splenocytes cultured without IL-2 showed no proliferative capacity, and cell viability decreased from 75.3% + 8.0% at day 1 to 8.8% 9.2% by day 3 of culture. No viable lymphocytes were recovered after 3 days in culture without IL-2. These data demonstrate maximal antifungal activity, antitumor activity, and cell viability after 7 days of culture with IL-2. This lymphocyte population was selected for further analysis. Competitive inhibition of antifungal and antitumor activity. To assess whether the biological activity of the IL-2-activated lymphocytes could be inhibited by both fungal and tumor cells, cold target inhibition studies were performed. In all experiments, homologous competitors provided maximum inhibition of their respective targets (Fig. 4). No competitive inhibition by naive thymocytes was observed in any experiment. Figure 4A depicts cold target inhibition of C. albicans. Both P815 and YAC-1 cells demonstrated the ability to compete for the activity of IL-2-activated splenocytes against C. albicans targets. The competitive inhibition observed with both tumor cell lines was significantly higher than that of naive thymocytes (P < 0.05 at the three highest ratios examined). The activity of the IL-2-activated lymphocytes for the YAC-1 tumor cell line was inhibited by both the P815 tumor cell line and to a lesser extent C. albicans. The inhibition by P815 cells and C. albicans was significant at the three highest ratios examined (P < 0.05) (Fig. 4B). The
activity of the IL-2-activated lymphocytes for the P815 cell line was almost completely inhibited by the YAC-1 cell line. C. albicans effectively competed for the activity of the IL-2-activated lymphocytes with the P815 tumor cell line at the three highest ratios examined (P < 0.05) (Fig. 4C). To ensure that the cold target competitors did not inhibit biological assessment through released cellular factors, culture supernatants from the competitor cell populations were collected and assessed for cytotoxic or growth-inhibitory activity. No effect was observed for any of the supernatants (data not shown). These data suggest that the IL-2-activated lymphocytes which interact with fungal targets also interact with P815 and to some extent YAC-1 targets. Susceptibility of various Candida strains to the antifungal activity. To determine whether the antifungal effect of the splenocytes was limited to the C. albicans strain (ATCC 58716) utilized in the experiments described above, the growth-inhibitory effect of the IL-2-activated lymphocytes was assessed for various clinical isolates of C. albicans, Candida tropicalis, Candida parapsilosis, and Torulopsis glabrata (Fig. 5). All isolates of C. albicans and other Candida species evaluated were susceptible to lymphocytemediated growth inhibition, indicating that the anti-C. albicans activity observed was not limited to C. albicans ATCC 58716. Species restriction of antifungal activity. Recent work with Cryptococcus neoformans suggests that even though murine lymphocytes inhibit that fungus, human peripheral blood lymphocytes do not seem to mediate antifungal activity
BENO AND MATHEWS
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proliferation and cell recovery from cultures of IL-2-activated splenocytes. Splenocyte populations were cultured for 0 to 9 days with 1,500 U of IL-2 per ml and assessed at daily intervals for antitumor activity against YAC-1 and P815 cells and anti-C. albicans activity. C. albicans growth inhibition was assessed by the incorporation of [3H]uridine. Data are presented as inhibitory units per 107 cells at 20% inhibition. YAC-1 and P815 cell cytotoxicity is expressed as lytic units per 107 cells at 20% cytotoxicity as judged by 51Cr release. Data are expressed as the means ± the standard deviations for three independent experiments.
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ANTIFUNGAL ACTIVITY OF IL-2-ACTIVATED SPLENOCYTES
VOL. 60, 1992
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104 105 10 10 5 X 106 X lO 104 1lo10B s x 106 1o6 COLD TARGETS COLD TARGETS COLD TARGETS FIG. 4. Unlabelled target competition studies with C. albicans (a), YAC-1 cells (0), and P815 cells (A). IL-2-activated lymphocytes were utilized at a 40:1 effector/target ratio for calculation of percent competitive inhibition. (A) Competitive inhibition of antifungal activity by C. albicans, YAC-1 cells, and P815 cells was assessed by the [3H]uridine method. (B) Competitive inhibition of YAC-1 cell cytotoxicity by C. albicans, YAC-1 cells, and P815 cells as assessed by 51Cr release. (C) Competitive inhibition of P815 cell cytotoxicity by C. albicans, YAC-1 cells, and P815 cells as assessed by 51Cr release. Unlabelled homologous target competitors are shaded. Unlabelled heterologous target competitors are unshaded. Thymocytes (K) were utilized as irrelevant competitors. Data are expressed as the means + the standard deviations for three independent experiments.
104
against cryptococci (25). Human peripheral blood mononuclear cells were activated with IL-2 for 7 days and then assessed for anti-C. albicans activity. As shown in Fig. 6, human peripheral blood mononuclear cells (plastic nonadherent lymphocytes after culture) mediated C. albicans growth inhibition. The antifungal activity was similar to that produced by human polymorphonuclear leukocytes in this assay. Little or no lymphocyte-mediated, antifungal activity was observed for mononuclear cells that were not stimulated with IL-2 (Fig. 6, No IL-2). These data suggest that the IL-2-induced, lymphocyte-mediated antifungal activity is not restricted to mice. DISCUSSION
Splenocytes derived from mice and immediately assessed for hyphal growth inhibition showed little or no activity. However, significant hyphal growth inhibition was observed when lymphoid cells were cultured with high concentrations of IL-2. Growth-inhibitory activity was mediated by nonadherent lymphocytes which had the capacity to lyse the NK-susceptible YAC-1 cell line and the NK-resistant P815 cell line. Other investigations have failed to demonstrate lymphocyte-mediated activity against C. albicans (5, 11). Those investigations involved either freshly isolated lymphocytes or lymphocytes activated for short periods of time with low concentrations of IL-2 (5, 11, 12). NK activity is associated with a morphological cell type, the large granular lymphocyte (28, 31). Large granular lymphocytes have been
isolated in high purity from discontinuous Percoll gradients (17). To enrich for NK activity, day 0 splenocytes were subjected to discontinuous Percoll separation. Low-density fractions showed a significant increase in NK activity compared with that of the unseparated splenocyte population. No increase in C. albicans growth inhibition was observed with the large granular lymphocyte-enriched cell population. These data are consistent with those of previous studies which showed no enrichment for C. albicans growth inhibition in cell populations enriched in NK activity (12, 40). Murine splenocytes activated with IL-2 for 24 h showed no significant increase in C. albicans-growth-inhibitory activity despite a significant increase in NK activity. These data are consistent with previous findings demonstrating that splenocytes activated for a short time period with IL-2 possessed an enhanced NK activity but could not mediate C. albicans growth inhibition (11). However, this study demonstrates that with extended culture time in high concentrations of IL-2, the capacity to inhibit the growth of C. albicans is acquired by lymphocytes and is associated with the capacity to lyse an NK-insensitive cell line. The appearance of optimal anti-C. albicans activity paralleled the development of anti-P815 cell activity. These data are consistent with our previous observations and extend that study to a complete analysis of the optimal time period in culture for maximal development of lymphocytes capable of mediating growth inhibition of C. albicans (7). In our previous study, lymph node cells were cultured for 3 days in IL-2. Such IL-2activated lymphocytes mediated growth inhibition of C.
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INFECT. IMMUN.
0-~o
134.5 ± 18.4 IU * 125.9 :t 14.2 IU 61.7 ±: 10.2 IU 138.2 ± 20.1 IU
C. albicans C. alblcans * A -A T. glabrata A- A C. albicans --
75 -
EJ- E C. tropicalls
z
0 50
+
136.1 ± 23.5 IU * --U C. parapallosls 135.5 ±: 18.8 IU V V C. tropicall. 122.2 ± 21.6 IU
m
1
Z-
z 25 + T
I
l 0
5
l0
20
50
E: T RATIO FIG. 5. Growth-inhibitory capacity of IL-2-activated lymphocytes for various Candida isolates and species. IL-2-activated lymphocytes were assessed for inhibition of growth of clinical isolates of C. albicans and other Candida spp. by the [3H]uridine method, and data are presented as percent inhibition and inhibitory units (IU) per 107 cells at 20% inhibition. *, C. albicans ATCC 58716. Data are expressed as the means + the standard deviations for three independent experiments. as well as lysed an NK-susceptible and to a lesser extent an NK-resistant cell line. Those data are compatible with our current findings. In this study, significant growth-
albicans
inhibitory activity for C. albicans appeared by the third day of culture and reached maximal activity by the seventh day of culture. The significant increases in activity for C. albicans and the NK-resistant target paralleled one another and were preceded by the rise in activity for the NK-sensitive target. In this investigation, we have shown that optimal growth and recovery of lymphocytes which mediate antifungal activity occur by the seventh day of culture, and that cell population has been selected for further evaluation. Extended culture time permitted not only the development of antifungal activity but also the development of broad tumor cytotoxic activity for NK-sensitive and -insensitive cell lines. The development of antifungal and antitumor activities may be a consequence of the expansion of IL-2responsive lymphocytes capable of mediating the antitumor activity. Previous studies have shown that IL-2 activation of lymphoid cultures results in a decrease in the number of IL-2-unresponsive lymphocytes, with a concomitant increase in the number of lymphocytes responsive to IL-2 (39). Studies have indicated that at least 3 days of IL-2 activation are required for the induction of anti-P815 cell activity, with 7 days required for maximal activity (23, 24). The results of this study do not demonstrate an absolute requirement for the expansion of an IL-2-activated cell population which mediates the C. albicans growth inhibition. However, Fig. 3 clearly shows that maximal antifungal and antitumor activi-
ties are preceded by an initial decrease in recoverable lymphocytes which is followed by marked cellular expansion. These results suggest that expansion of fungus- and/or tumor-reactive cell populations results in a concomitant increase in biological activity for each target cell type. The results are consistent with a lymphocyte population mediating both anti-C. albicans and anti-P815 cell activities. Heterologous cold targets inhibited biological activity for either fungal or tumor targets in a competitive and reciprocal manner, suggesting that the IL-2-activated lymphocytes possess the ability to interact with all three targets. A directed interaction between effector and target is likely in that supernatants taken from cultures of IL-2-activated lymphocytes had no effect on any target examined. However, it is possible that a soluble substance released by lymphocytes upon contact with fungi is responsible for growth inhibition. The extent of competition of IL-2-activated lymphocytes for C. albicans was greater for P815 than for YAC-1 cells, suggesting that the IL-2-activated lymphocytes which interact with C. albicans are more reactive with P815 than with YAC-1 cells. The greater competition by P815 cells might not necessarily reflect variation in target surface but might rather be a characteristic of the IL-2activated lymphocytes. The IL-2-activated lymphocytes which mediate C. albicans growth inhibition might be more adhesive for P815 than for YAC-1 cells. The reciprocal inhibition of anti-YAC-1 cell activity by C. albicans indicates recognition of C. albicans by a portion of the IL-2activated lymphocyte population which mediates activity
ANTIFUNGAL ACTIVITY OF IL-2-ACTIVATED SPLENOCYTES
VOL. 60, 1992
*-*
PMN
0-
IL-2
A 75
-
50
4-
A
861
No IL-2
z 0
m
T
m z
--T
25 + T
C
1
1
T
A
I
A
1
±L
0
5
10
20
50
E : T RATIO FIG. 6. Growth-inhibitory capacity of IL-2-activated human lymphocytes for C. albicans. IL-2-activated lymphocytes and polymorphonuclear leukocytes were assessed for C, albicans growth inhibition by the [3H]uridine method, and data are presented as percent inhibition. Data are expressed as the means the standard deviations for three independent experiments.
against YAC-1 cells. The inhibition is not complete, suggesting the possibility that other IL-2-activated lymphocytes, besides those reactive with C. albicans, may interact with YAC-1 cells. The interaction of IL-2-activated lymphocytes with tumor targets is not as easily inhibited with C. albicans as with tumor competitors. The incomplete cold target inhibition observed by heterologous targets may be explained by the heat inactivation of C. albicans. Heat alteration of receptors may lower the activity of lymphocytes for hyphae (8). The mechanism of IL-2-activated lymphocyte interaction with C. albicans may require a long period of time, and once bound, IL-2-activated lymphocytes may not dissociate from the cell surface and locate to another target as quickly as with tumor cells. NK-cryptococcus interactions require 2 h for initiation of antifungal activity, while NK-mediated YAC-1 cell cytotoxicity requires 20 min (27). Furthermore, NK-cryptococcus conjugate formation is smaller, does not appear to interdigitate, and is generally much less intimate than is the broad area of interdigitating membrane contact observed in NK-tumor cell conjugates (18). The interaction of the IL-2-activated lymphocytes with C. albicans might also require a longer time period for initiation of antifungal activity, leading to the reduced competitive inhibition observed with C. albicans. The inhibition of biological activity by heterologous tar-
gets is statistically significant and demonstrates that both C. albicans and tumor cell lines competitively and reciprocally inhibit the activity of the IL-2-activated lymphocyte population. Previous studies of fungal interactions with both cryptococci (18, 29) and C. albicans (40) have demonstrated the ability of fungi to competitively inhibit lymphocytemediated biological activity. The apparent interaction of IL-2-activated lymphocytes with all three targets, combined with the observed biological activity for all three targets, suggests broad recognition and reactivity of the IL-2-activated lymphocytes. Whether the IL-2-activated lymphocytes are a single homogeneous cell population or are composed of more than one subpopulation is unknown at this time. This study has focused on the biological activity of lymphocytes cultured for 7 days with IL-2 because of the enhanced activity that cell population exhibits for C. albicans. Whether that cell population is the same as the one that mediates anti-C. albicans activity after 3 days of culture with IL-2 is unclear but will be the subject of other investigations. This study shows that the biological activity of the IL-2activated lymphocyte population which expresses maximal antifungal activity is neither an NK or short-term-activated NK population, because those cell populations failed to
862
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mediate C. albicans growth inhibition. The IL-2-activated lymphocytes are clearly lymphokine activated, but whether they are a lymphokine-activated killer lymphocyte (LAK) population or a subset of the LAK population is unclear. Various studies have revealed substantial heterogeneity in LAK populations following activation with IL-2 (24, 26, 32). The lytic activity of various types of LAK and their apparent reactivities are highly dependent on their states of activation. No previous studies have demonstrated C. albicans growth inhibition or antifungal activity for LAK populations. It is also clear that the anti-C. albicans activity is not limited to a single fungal strain, in that a number of clinical isolates of the fungus as well as closely related fungi were susceptible to growth inhibition mediated by the IL-2-activated lymphocytes. This study is consistent with previous investigations (7, 11, 36) but demonstrates that increased concentrations of IL-2 and more extended culture periods are required to obtain a highly activated lymphocyte population capable of affecting the growth of the fungus. Whether this phenomenon functions in vivo, as a form of host defense, has yet to be established. It is possible that the elaboration of cytokines such as IL-2 and the concomitant induction of activated lymphocytes indeed serve as a form of host defense against C. albicans infection. Protection of the host by this form of immunological effector may at least in part account for the resistance of the C57BL/6 mouse strain to challenge with the microorganism. The experiments described herein do not address the issue of differences among inbred strains of mice in their resistance to C. albicans. Rather, this investigation has centered on the antifungal activity of lymphocytes derived from the C57BL/6 mouse strain, which is highly resistant to C. albicans. Analysis and comparisons of other murine strains with the C57BL/6 strain will be the subject of future investigations. However, IL-2-activated lymphocytes may serve as potential effectors during the development of fungal infections and may be considered to be possible therapeutic means to limit fungal infections in immunocompromised individuals. ACKNOWLEDGMENTS This research was supported by a grant from the Cancer Federation and the American Cancer Society, Inc. (grant IM-489). We are grateful to the Hoffmann-LaRoche Corporation for supplying IL-2. REFERENCES 1. Ashman, R. B. 1990. Murine candidiasis: cell-mediated immune responses correlate directly with susceptibility and resistance to infection. Immunol. Cell Biol. 68:15-20. 2. Ashman, R. B., and J. M. Papadimitriou. 1987. Murine candidiasis. Pathogenesis and host responses in genetically distinct inbred mice. Immunol. Cell Biol. 65:163-171. 3. Ausiello, C., A. Maleci, G. C. Spagnoli, G. Antonelli, and A. Cassone. 1988. Cell-mediated cytotoxicity in glioma-bearing patients: differential responses of peripheral blood mononuclear cells to stimulation with interleukin-2 and microbial antigen. J. Neuro-Oncol. 6:329-338. 4. Baccarini, M., F. Bistoni, and H. L. Lohmann-Matthes. 1985. In vitro natural cell-mediated cytotoxicity against Candida albicans: macrophage precursors as effector cells. J. Immunol. 134:658-665. 5. Baccarini, M., F. Bistoni, P. Puccetti, and E. Garaci. 1983. Natural cell-mediated cytotoxicity against Candida albicans induced by cyclophosphamide: nature of the in vitro cytotoxic effector. Infect. Immun. 42:1-9. 6. Baccarini, M., A. Vecchiarelli, A. Cassone, and F. Bistoni. 1985. Killing of yeast, germ-tube and mycelial forms of Candida albicans by murine effectors as measured by a radiolabel release
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microassay. J. Gen. Microbiol. 131:505-513. 7. Beno, D. W. A., and H. L. Mathews. 1990. Growth inhibition of Candida albicans by interleukin-2 induced lymph node cells. Cell. Immunol. 128:89-100. 8. Bouchara, J.-P., G. Tronchin, V. Annaix, R. Robert, and J.-M. Senet. 1990. Laminin receptors on Candida albicans germ tubes. Infect. Immun. 58:48-54. 9. Diamond, R. D., and C. C. Haudenschild. 1981. Monocytemediated serum-independent damage to hyphal and pseudohyphal forms of Candida albicans in vitro. J. Clin. Invest. 67:173182. 10. Diamond, R. D., and R. Krzesicki. 1978. Mechanisms of attachment of neutrophils to Candida albicans pseudohyphae in the absence of serum, and of subsequent damage to pseudohyphae by microbicidal processes of neutrophils in vitro. J. Clin. Invest. 61:360-369. 11. Djeu, J. Y., and K. Blanchard. 1987. Regulation of human polymorphonuclear neutrophil (PMN) activity against Candida albicans by large granular lymphocytes via release of PMNactivating factor. J. Immunol. 139:2761-2767. 12. Djeu, J. Y., K. Blanchard, A. L. Richards, and H. Friedman. 1988. Tumor necrosis factor induction by Candida albicans from human natural killer cells and monocytes. J. Immunol. 141:4047-4052. 13. Goto, M., and N. J. Zvaifler. 1985. Characterization of the natural killer-like lymphocytes in synovial fluid. J. Immunol. 134:1483-1486. 14. Grimm, E. A., A. Mazumder, H. Z. Zhang, and S. A. Rosenberg. 1982. Lymphokine-activated killer cell phenomenon. Lysis of natural killer-resistant fresh solid tumor cells by interleukin 2-activated autologous human peripheral blood lymphocytes. J. Exp. Med. 155:1823-1841. 15. Hashimoto, T. 1983. A micromethod for determination of the viability of Candida albicans hyphae. J. Microbiol. Methods 1:89-98. 16. Herberman, R. B., and H. T. Holden. 1978. Natural cellmediated immunity. Adv. Cancer Res. 27:305-377. 17. Hidore, M. R., and J. W. Murphy. 1986. Correlation of natural killer cell activity and clearance of Cryptococcus neofornans from mice after adoptive transfer of splenic nylon wool-nonadherent cells. Infect. Immun. 51:547-555. 18. Hidore, M. R., N. Nabavi, C. W. Reynolds, P. A. Henkart, and J. W. Murphy. 1990. Cytoplasmic components of natural killer cells limit the growth of Cryptococcus neofornans. J. Leukocyte Biol. 48:15-26. 19. Lafreniere, R., M. S. Rosenstein, and S. A. Rosenberg. 1986. Optimal methods for generating expanded lymphokine activated killer cells capable of reducing established murine tumors in vivo. J. Immunol. Methods 94:37-46. 20. Lanier, L. L., A. M. Le, M. R. Ldoken, J. H. Phillips, and C. I. Civin. 1987. The relationship of CD16 (Leu-11) and Leu-19 (NKH-1) antigen expression of human peripheral blood NK cells and cytotoxic T lymphocytes. J. Immunol. 136:4480-4486. 21. Mahanty, S., R. A. Greenfield, W. A. Joyce, and P. W. Kincade. 1988. Inoculation candidiasis in a murine model of severe combined immunodeficiency syndrome. Infect. Immun. 56: 3162-3166. 22. Marquis, G., S. Montplaisir, M. Pelletier, S. Mousseau, and P. Auger. 1986. Strain-dependent differences in susceptibility of mice to experimental candidosis. J. Infect. Dis. 154:906-909. 23. Merluzzi, V. J. 1985. Comparison of murine lymphokine-activated killer cells, natural killer cells, and cytotoxic T lymphocytes. Cell. Immunol. 95:95-104. 24. Merluzzi, V. J., M. D. Smith, and K. Last-Bareny. 1986. Similarities and distinctions between natural killer cells and lymphokine activated killer cells. Cell. Immunol. 100:563-569. 25. Miller, M. F., T. G. Mitchell, W. J. Storkus, and J. R. Dawson. 1990. Human natural killer cells do not inhibit growth of Cryptococcus neoformans in the absence of antibody. Infect. Immun. 58:639-645. 26. Mule, J. J., J. A. Krosnick, and S. A. Rosenberg. 1989. IL-4 regulation of murine lymphokine activated killer activity in vitro. Effects on the IL-2 induced expansion, cytotoxicity, and
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