A quantitative radiometric assay to measure mammalian cell binding to hyphae of Candida albicans

Journal of Immunological Methods, 165 (1993) 113-119

113

© 1993 Elsevier Science Publishers B.V. All rights reserved 0022-1759/93/$06.00

JIM06811

A quantitative radiometric assay to measure mammalian cell binding to hyphae of Candida albicans Christopher B. Forsyth and Herbert L. Mathews Department of Microbiology and Immunology, Stritch School of Medicine, Loyola University of Chicago, Maywood, IL 60153, USA (Received 5 February 1993, revised received 7 May 1993, accepted 3 June 1993)

A rapid and reproducible assay has been developed to measure the capacity of lymphocytes to bind to Candida albicans. Lymphocytes that bound to C. albicans were either the large granular lymphocyte cell line, YT, or interleukin-2 activated lymphocytes. Lymphocyte binding was assessed as the associated radioactivity of 51Cr-labeled lymphocytes with preformed hyphae. The assay was sensitive to detection of 0.6 lymphocytes/one hyphal form at one half maximal lymphocyte binding capacity. The assay correlated well with direct microscopic assessment of lymphocyte binding to C. albicans and provided quantitative radiometric data. Although the assay was developed for the assessment of lymphocyte adhesion to C. albicans, it can be used to measure binding of other mammalian ceils (e.g., polymorphonuclear leukocytes) to this fungus. In addition, the assay may be used to identify molecules involved in the adhesion of lymphocytes and other mammalian cells to C. albicans. Key words: Candida albicans; Adhesion; Lymphocyte; YT

Introduction

Methodologies have been reported by which to assess the in vitro binding of C. albicans to mam-

Correspondence to." H.L. Mathews, Department of Microbiology and Immunology, Stritch School of Medicine, Loyola University of Chicago, Maywood, IL 60153, USA. Tel.: (708) 216-4586; Fax: (708) 216-9574. Experimental animal usage was in accordance with institutional guidelines. Informed consent was obtained from human subjects after explanation. Abbreviations: DMEM, Dulbecco's minimal essential medium; FBS, fetal bovine serum; HBSS, Hanks' balanced salt solution; IAL, interleukin-2 activated lymphocytes; IL-2, interleukin-2; i.p., intraperitoneal; LGL, large granular lymphocyte; MASH, multiple automated sample harvester; PMN, polymorphonuclear leukocyte; SDA, Sabouraud's dextrose agar; 2-ME, 2 mercaptoethanol.

malian tissues (Calderone and Braun, 1991), cells (Sandin et al., 1987; Rotrosen et al., 1985; Sobel et al., 1981), extracellular matrix (Klotz and Smith, 1991) and spleen and lymph node nonendothelial sites (Cutler et al., 1990). Fundamental to each assessment has been the microscopic enumeration of fungal binding to mammalian cells or proteins. We have developed an alternative means by which to accurately and simply assess mammalian cell (specifically lymphocyte) binding to the hyphal form of C. albicans. This procedure is an adaptation of previously published methodologies (Dustin and Springer, 1989; Van Seventer et al., 1991). However, the procedure reported herein takes advantage of the capacity of C. albicans to bind to plastic surfaces (Kennedy et al., 1989; Hazen, 1989) which allows for easy harvest of the hyphae associated lymphocytes.

114

Therefore, large numbers of experimental sampies can be processed with ease. The short period of the assessment and the reproducibility of the assay ensure a rapid method for the in vitro measurement of mammalian cell binding to this fungus.

Materials and methods

Mice C57B1/6 and B A L B / c female mice, ages 6-7 weeks, were obtained from Harlan, Indianapolis, IN. Mice were 6-12 weeks of age when used in experiments. B A L B / c mice were used solely for the preparation of monoclonal antibody containing ascites fluid. Fungal culture Candida albicans American Type Culture Collection (ATCC) 58716, obtained from Dr. T. Hashimoto, Loyola University of Chicago, Maywood, IL, was used throughout this investigation. Cultures were stored at 25°C on Sabouraud's dextrose agar (SDA) (Becton Dickinson, Cockeysville, MD). Cells used for experimentation were cultured overnight at 37°C on SDA, collected as isolated colonies, and washed once in Hanks' balanced salts solution (HBSS). Yeast cultures were enumerated microscopically and those with greater than 15% budding were discarded. Clinical isolates of C. albicans were obtained from the Clinical Microbiology Laboratories of the Loyola University Medical Center, Maywood, IL. LGL-like cell line YT Human leukemia, large granular lymphocyte (LGL)-like YT cells were originally obtained from E. Kovacks, Loyola University of Chicago, Maywood, IL. A subline of these original cells was selected in our laboratory for its ability to bind to C. albicans hyphae. These YT cells were cultured at 5 × 104 cells/ml in Falcon 24-well plates (Becton Dickinson, Lincoln Park, NJ) in RPMI 1640 media supplemented with 10% fetal bovine serum (JRH Bioscience, Lenexa, KS), 5 × 10 -s M 2mercaptoethanol (2-ME), 100 U penicillin and 100 /xg streptomycin/ml, 0.1 mM nonessential

amino acids, and 2 mM L-glutamine (all from Gibco, Grand Island, NY). YT cells were passaged every 2 days in this medium with the addition of 2.5% 2-day YT culture conditioned medium.

IL-2 activation of lymphocytes Mouse splenocytes were placed in culture medium containing 5 × 10 -s M 2-ME at a concentration of 2.5 × 10 6 cells/ml with 1500 U / m l interleukin-2 (IL-2) (Hoffman-La Roche, Nutley, N J) in Falcon Multiwell plates (Becton Dickinson, Lincoln Park, N J) for 7 days. This concentration of IL-2 was determined to be optimal. The cells were harvested following incubation at 37°C, overlaid onto lymphocyte separation medium (Litton Bionetics, Kensington, MD) and centrifuged at 1000 x g for 20 min. The cells at the interface were washed twice with HBSS prior to assessment of binding activity. These lymphoid ceils were > 99% lymphocytes as judged by Wright-Giemsa staining. Mammalian cells Mouse polymorphonuclear leukocytes (PMN) were elicited by intraperitoneal (i.p.) injection of 1.0 ml thioglycollate broth (Difco Labs, Detroit, MI). 3 h later the peritoneal cavity was washed with 10 ml of HBSS, the elicited ceils enumerated and placed in culture medium at a concentration of 2.5 × 106 cells/ml in multiwell plates as described above for 18 h at 37°C and 5% CO 2. Non-adherent cells were recovered and were found to be greater than 90% viable PMN as judged by vital dye exclusion (0.04% trypan blue) and Wright-Giemsa staining. PMN culture conditions were similar to those described previously (Kurita et al., 1991; Faried et al., 1993). Human erythrocytes were obtained from peripheral blood and were greater than 99.99% erythrocytes as judged by Wright-Giemsa staining. The EL-4 and NYC tumors are maintained routinely in this laboratory. 51Cr labeling of mammalian cells 100 /xCi of SlCr (New England Nuclear, Boston, MA) were added to 1 × 107 mammalian cells in a final volume of 0.2 ml of HBSS. The cells were incubated at 37°C with 5% CO 2 for 1 h

115

with agitation every 10 rain, washed three times in HBSS and enumerated with a hemocytometer.

Binding assay This assay is an adaptation of previously described procedures which utilize 51Cr-labeled cells to quantify cellular binding to substrate (Dustin and Springer, 1989; Van Seventer et al., 1991). C. albicans hyphae were prepared by growth in RPMI 1640 for 3 h at 37°C in flat-bottomed 96-well plastic plates (Corning: #25861-96). After 3 h 90-100% confluence of hyphae was obtained when 1 x 105 yeast were delivered initially to each well of these assay plates. This inoculum produced 100% hyphal forms ranging in length from 30-50 lzm. 5~Cr-labeled mammalian cells were added to individual wells of the assay plates and incubated in a 5% CO~ incubator at 37°C for 1 h. The assay was terminated by removal of unbound mammalian cells from each well, either with a pasteur pipet with subsequent 3 x wash with 200 Izl of HBSS, or by use of a multiple automated sample harvester (MASH) (PHD cell harvester, Cambridge Scientific, Cambridge, MA). When the MASH was used the assay wells were washed 3 x with 200 /zl of saline. After either wash procedure 200 /zl of 0.5% NP-40 (Sigma Chem. Corp., St. Louis, MO) was added to each well for 20 rain. In both cases the 0.5% NP-40 containing supernates were removed with a pasteur pipet and associated radioactivity determined. Results are expressed as percentage cells bound as judged by the associated SlCr as follows: (experimental cpm) - (background cpm) % bound

(maximum cpm) - (background cpm)

x 100

Maximum cpm release was obtained by adding 0.5% NP40 directly to mammalian cells. Experimental means were calculated from triplicate values.

Inhibition of binding assay These experiments followed the protocol for the binding assay, except that 5 X 10 4 radiolabeled YT cells were combined in different proportions with either unlabeled YT or unlabeled mouse thymocytes in 200 /~1 HBSS. The inhibi-

tion assay was then completed as described above for the binding assay. Results are expressed as percentage inhibition of ceils bound to hyphae as judged by associated 51Cr as follows: (experiment c p m - b a c k g r o u n d cpm) % inhibition = 1 -

(maximum cpm - background cpm)

x 100

Maximum cpm release was obtained by adding 0.5% NP-40 directly to radioactively labeled YT cells. Experimental means were calculated from triplicate values.

Monoclonal antibody Hybridoma cells LM2/1.6.11 and TS2/18 were obtained from the Developmental Studies Hybridoma Bank maintained by the Johns Hopkins University School of Medicine, Baltimore, MD, and the University of Iowa, Iowa City, IA, under contract N01-HD-6-2915 from the NICHD. P3X63-Ag8.653 was obtained from C. Lange, Loyola University of Chicago, Maywood, IL. Antibody employed in this study was mouse ascites fluid from i.p. passaged cells.

Results

Several mammalian cell populations were assayed for their ability to bind the hyphal form of C. albicans (Fig. 1). Optimal binding to hyphae was achieved with the human LGL-like cell line YT and with murine IL-2 activated lymphocytes (IAL). 15% of maximum binding was achieved w i t h ] 0 4 cells, 50% of maximum with 6 x 10 4 cells and maximum values between 1 x 105 and I x 106 cells per assay well. No apparent binding to hyphae was observed with human erythrocytes, murine thymocytes, murine splenocytes, the murine T cell lymphoma EL-4 or the murine B cell leukemia NYC. Visual inspection of the assay wells revealed hyphae bound lymphocytes prior to the addition of NP-40. Associated radioactivity correlated visually with the number of lymphocytes bound to the hyphae. Few or no lymphocytes appeared to adhere to the plastic of the assay well surface. Associated radioactivity for assay wells that contained no C. albicans (but did

116

contain radiolabeled lymphocytes) was always less than 5% of the total counts per minute added. These data were used as background values. Data not shown. The conditions employed for these assessments were determined to be optimal. In the development of this assay procedure comparisons were made of methods for immobilization of C. albicans, C. albicans initial cell concentration, degree of C. albicans confluence after culture, time and temperature of lymphocyte interaction with C. albicans, and the culture medium in which the binding assay was performed. 12 × 75 borosilicate glass and polystyrene plastic tubes did not provide the surface necessary for reproducible immobilization of C. albicans. The flat surface of the 96 well cluster plates provided highly reproducible results and was simpler and easier to manipulate. Comparisons of varying cell numbers of C. albicans (5 x 104-2 X 105) immobilized to the plastic well surfaces showed that maximum binding to the fungal hyphae was achieved by incubation of 1 x 105 yeasts/assay well and incubating at 37°C and 5% CO 2 for 3 h. This initial yeast cell number produced approximately 100% hyphal confluency during the assay with hyphal interdigitation across the entire surface of each well. Maximal binding occurred at 60 min of incubation with shorter periods of time resulting

100

/ T?--?T

U

75 2 z

50~25

104

1O5 106 CELL NUMBER

10"7

Fig. 1. Binding of various cell types to C. albicans (1 x 1 0 5 / well). The binding of different 51Cr-labeled cell types to C. albicans was assessed by the retention of 51Cr-labeled cells. Data are presented as mean %bound + the standard deviation (SD). YT, © o ; IAL, • , ; mouse PMN, • o; h u m a n e r y t h r o c y t e s , zx A ; EL-4, [] D ; NYC, A - - A .

100

75 ~ 50

/ / T//

0 l

105

106 107 108 CELL NUMBER Fig. 2. Competition by non-radiolabeled cell types for 51Crlabeled YT cell binding to C. albicans. Binding of YT cells to C. albicans was assessed by the retention of 5lCr-labeled cells in the presence of YT cells or thymocytes. D a t a are presented as mean %bound_+SD. YT, o o; mouse thymocytes, • 0.

in less lymphocyte association with hyphae. No difference in lymphocyte binding was observed at 25°C or 37°C. Experiments that compared YT binding to C. albicans hyphae in either HBSS, RPMI 1640, or RPMI 1640 with either 0.1% or 1.0% FBS were performed. There was no difference in binding between HBSS and RPMI 1640, however those wells containing FBS averaged (from 10 to 50%) fewer bound YT cells. Data not shown. To further establish the YT cell interaction with C. albicans hyphae, non-radioactively labeled YT and thymocytes were used to compete for the binding of radioactively labeled YT to the fungal surface (Fig. 2). No competitive binding of radioactively labeled YT was seen with non-radioactive thymocytes. Non-radioactive YT effectively competed for radioactive YT binding to hyphae. Inspection of assay wells revealed no horn•typic aggregation of YT cells. Another important aspect of this assay is the use of a multiple automated sample harvester (MASH) to wash and remove unbound mammalian cells. In a representative experiment 'hand washing' with a pasteur pipet was compared to the use of the MASH (Table I). Not only is the MASH more simplistic and easy to use but also the MASH results in less variability as judged by comparison of standard deviation for the MASH versus that for the hand washing.

117 TABLE I COMPARISON OF HAND-WASHING AND MULTIPLE AUTOMATED SAMPLE HARVESTER RECOVERY OF RADIOACTIVITY ASSOCIATED WITH LYMPHOCYTES BOUND TO C. ALBICANS. Lymphocyte cell number a

MASH b

Hand washing

Mean cpm _+SD Mean cpm _+SD 1.0× 105 0.8×105 0.6×105 0.4× 105

that six clinical isolates of the microorganism were bound by YT in a similar if not identical manner as was strain ATCC 58716. Data not shown.

88,008 3,974 75,142 1,446 49,103 2,272 37,222 2,822

80,536 66,771 48,030 39,164

7,092 4,203 3,820 3,737

a YT cells were radiolabeled with 51Cr. b MASH, multiple automated sample harvester.

In order to determine the assay's potential utility in the analysis of molecules involved in lymphocyte adhesion, antibody containing ascites was diluted serially and preincubated with YT cells on ice (Fig. 3). Ascites containing anti-CD11b inhibited the binding of lymphocytes to C. albicans. The level of inhibition obtained with antiC D l l b was markedly greater than that achieved with either ascites containing isotype matched anti-CD2 or ascites produced from the P3-X63Ag8.653 fusion partner. C D l l b and CD2 are expressed by the YT cell line (Springer et al., 1987; Diamond and Springer, 1993). The binding assay described herein is not restricted to a particular strain of C. albicans in

100-

75 3 50

25

~o 0

I

I

J

I

I

I

J

I

I

100

200

300

400

500

600

700

800

900

1000

RECIPROCAL DILUTION OF ASCITES

Fig. 3. Inhibition of YT cell binding to C. albicans by use of monoclonal antibody. Data are expressed as mean % inhibition _4_SD of YT cell binding in the presence of mouse ascites fluid from hybridoma LM2/1.6.11 (anti-CD llb), o o; TS2/18 (anti-CD2), z x - - L x ; or P3-X63Ag8.653, • •.

Discussion

It has been the purpose of this study to develop an efficient and convenient radiometric assay for the quantitative measurement of the adhesion of lymphocytes to the hyphal form of C. albicans. Many assays have been reported to assess the binding of C. albicans to mammalian cells and tissues (Calderone and Braun, 1991; Sandin et al., 1987; Cutler et al., 1991). Many fewer assays have been devised to measure the reaction of mammalian cells with fungi. These assays have employed the microscopic enumeration of the interaction of natural killer lymphocytes with Cryptococcus neoformans (Hidore and Murphy, 1989; Nabavi and Murphy, 1985). Those investigators demonstrated the direct interaction of natural killer cells with C. neoformans as judged by both light microscopy as well as scanning electron microscopy. Conjugate formation between lymphocytes and C. albicans has been demonstrated by flow cytometry (Zunino and Hudig, 1988). Indirect evidence for the interaction of natural killer cells and IL-2 activated lymphocytes has been shown by the competitive inhibition of functional activity of these cell types for their known targets (Zunino and Hudig, 1988; Beno and Mathews, 1992). Although useful, these assays do not necessarily lend themselves to a quick and direct assessment of the interaction of mammalian effectors with their targets. Therefore, this investigation has sought to identify a quantitative, radiometric assay to measure the adhesion of mammalian cells with C. albicans hyphae. The development of an optimal binding assay to measure adhesion of lymphocytes to C. albicans requires the consideration of a number of parameters including the identification of optimal numbers of C. albicans for maximal hyphae binding by lymphocytes, maximal concentration of mammalian effector cells, and the quantitative recovery of effector cell populations. The primary concern of this study was to develop an accurate

118 and usable assay that permitted daily experimental completion. Therefore each of these parameters was optimized with regard to this overall objective. As a consequence an essentially linear relationship between lymphocyte binding to C. albicans hyphae and associated radioactivity was achieved between 2.5 × 10 4 and 1 × 105 lymphocytes (Fig. 1). Such a relationship is not essential to measure the binding of lymphocytes to hyphae. However, such a relationship can be used to define interactions between mammalian cell populations and C. albicans and to determine lymphocyte surface molecules involved in interaction with the fungal surface (Figs. 2 and 3). By use of competitive assay within this linear range, slight changes in the number of lymphocytes bound to hyphae result in dramatic changes in the associated radioactivity. Such a sensitive and quantitative assay should be useful to identify cell populations and precise surface structures that mediate mammalian cell adhesion to the fungus. This assay was developed to measure the binding of lymphocytes to hyphae but the binding of PMN to hyphae can also be quantified (Fig. 1). The binding of PMN to hyphae was similar to the sigmoidal shaped binding characteristic of the YT and IL-2 activated lymphocyte population. The absolute differences in binding characteristics for the three cell populations may be due to the development of the binding assay for lymphocytes. It is possible that optimal binding conditions for PMN may vary slightly from that of the two lymphocyte populations investigated. The PMN employed in this study were induced with thioglycollate broth and cultured for 18 h before analysis of binding to C albicans. No significant change in viability of the PMN was noted and the PMN cultured for 18 h were more capable of binding C. albicans than freshly isolated thioglycollate-induced PMN. Other investigations have shown thioglycollate to inhibit the anti-fungal activity of mouse PMN (Brummer et al, 1985; Brummer et. al., 1986). The differential effect of thioglycollate broth upon mammalian cell binding to C. albicans has not been investigated. However, it has been the purpose of this study to develop an assay to measure lymphocyte binding to C. albicans. It is clear that mammalian cells (thymocytes, splenocytes, erythrocytes and two

tumor cell types) have no capacity to bind to C. albicans hyphae as judged by this assay. Likewise, it is apparent that a large granular lymphocyte cell line and IL-2 activated lymphocytes do bind to C. albicans hyphae and that such binding can be quantified. One of the potentially useful aspects of the developed assay may be the identification of surface molecules involved in the adhesion of mammalian ceils to C. albicans. Monoclonal antibodies reactive with YT surface molecules can inhibit the interaction of the lymphocytes with the surface of the fungi (Fig. 3). Such an inhibition of binding was not seen with ascites fluid derived from a hybridoma producing anti-CD2. The sensitive and quantitative nature of the binding assay should permit the full analysis of the surface molecules of lymphocytes that mediate adhesion of these mammalian cells to C. albicans hyphae. The assay described herein requires a short period of time to assess the binding of mammalian cells to fungal hyphae. The procedure utilizes a multiple automated sample harvester thus permitting rapid and reproducible assessment of large sample numbers. Finally, the assay correlates with visual inspection of the physical interaction of mammalian effectors with the hyphal surface. As such this assay procedure is an efficient, convenient, quantitative and correlative means by which to measure mammalian cell binding to C. albicans. In summary, the assay procedure described herein should be a useful means by which to assess mammalian cell adhesion to C. albicans in the hyphal form.

Acknowledgements This research was supported by PHS grant # AI31127. We are grateful to Hoffman-LaRoche Corporation for supplying IL-2.

References Beno, D.W.A. and Mathews, H.L. (1992)Growth inhibition of Candida albicans by IL-2 induced splenocytes. Infect. Immun. 60, 853. Brummer, E., Sugar, A.M. and Stevens, D.A. (1985) Enhanced oxidative burst in immunologicallyactivated but

119 not elicited polymorphonuclear leukocytes correlates with fungicidal activity. Infect. Immun. 49, 396. Brummer, E., McEwen, E.G. and Stevens, D.A. (1986) Fungicidal activity of murine inflammatory neutrophils: comparison with murine peripheral blood PMN. Clin. Exp. Immunol. 66, 681. Calderone, R.A. and Braun, P C. (1991) Adherence and receptor relationships in Candida albicans. Microbiol. Rev. 55, 1. Cutler, J.E., Brawner, D.L., Hazen, K.C. and Jutila, M.A. (1990) Characteristics of Candida albicans adherence to mouse tissue. Infect. Immun. 58, 1902. Diamond, M.S. and Springer, T.A. (1993) A subpopulation of Mac-1 ( C D l l b / C D 1 8 ) molecules mediates neutrophil adhesion to ICAM-1 and fibrinogen. J. Cell Biol. 120, 545. Dustin, M.L. and Springer, T.A. (1989) T-cell receptor crosslinking transiently stimulates adhesiveness through LFA-1. Nature 341,619. Faried, H.F., Tachibana, T. and Okuda, T. (1993) The secretion of the third component of complement (C3) by human polymorphonuclear leucocytes from both normal and systemic lupus erythematosus cases. Scand. J. Immunol. 37, 19. Hazen, K.C. (1989) Participation of yeast cell surface hydrophobicity in adherence of Candida albicans to human epithelial cells. Infect. Immun. 57, 1894. Hidore, M.R., Nabavi, N., Reynolds, C.W., Henkart, P.A. and Murphy, J.W. (1990) Cytoplasmic components of natural killer cells limit the growth of Cryptococcus neoformans. J. Leuk. Biol. 48, 15. Kennedy, M.J., Rodgers, A.L. and Yancey, Jr., R.J. (1989) Environmental alteration and phenotypic regulation of Candida albicans adhesion to plastic. Infect. Immun. 57, 3876. Klotz, S.A. and Smith, R.L. (1991) A fibronectin receptor on

Candida albicans mediates adherence of the fungus to extracellular matrix. J. Infect. Dis. 163, 604. Kurita, N., Brummer, E., Yoshida, S., Nishimura, K. and Miyaji, M. (1991) Antifungal activity of murine polymorphonuclear neutrophils against Histoplasma capsulatum. J. Med. Vet. Mycol. 29, 133. Nabavi, N. and Murphy, J.W. (1985) In vitro binding of natural killer cells to Cryptococcus neoformans targets. Infect. Immun. 50, 50. Rotrosen, D., Calderone, R.A. and Edwards, J.E. (1986) Adherence of Candida species to host tissues and plastic. Rev. Infect. Dis. 8, 73. Sandin, R.L., Rogers, A.L., Patterson, R.J. and Beneke, E.S. (1982) Evidence for mannose-mediated adherence of Candida albicans to human buccal cells in vitro. Infect. Immun. 35, 79. Sobel, J.D., Myers, P.G., Kaye, D. and Levison, M.E. (1981) Adherence of Candida albicans to human vaginal and buccal epithelial cells. J. Infect. Dis. 143, 76. Springer, T.A., Dustin, M.L., Kishimoto, T.K. and Marlin, S.D. (1987) The lymphocyte function-associated LFA-1, CD2, and LFA-3 molecules: cell adhesion receptors of the immune system. Ann. Rev. Immunol. 5, 223. Van Seventer, G.A., Newman, W., Shimizu, Y., Nutman, T.B., Tanaka, Y., Horgan, K.J., Gopal, T.V., Ennis, E., O'Sullivan, D., Grey, H. and Shaw, S. (1991) Analysis of T cell stimulation by superantigen plus major histocompatibility complex class II molecules or by CD3 monoclonal antibody: costimulation by purified adhesion ligands VCAM-1, ICAM-I, but not ELAM-1. J. Exp. Med. 174, 901. Zunino, S.J. and Hudig, D. (1988) Interactions between human natural killer (NK) lymphocytes and yeast cells: human NK cells do not kill Candida albicans although C. albicans blocks NK lysis of K562 cells. Infect. Immun. 56, 564.