Human secondary immune responses induced by influenza in a serum-free tissue culture system

Journal oflmmunological Methods, 82 (1985) 333-339

333

Elsevier JIM03608

Human Secondary Immune Responses Induced by Influenza in a Serum-Free Tissue Culture System 1 John M. Weiler

2,3, Stephen B. Rinderknecht,

Murali D. Sharath and

Robert Yarchoan Department of Internal Medicine, lowa City Veterans Administration Medical Center, The University of Iowa, Iowa City, IA 52242, and the National Cancer Institute, National Institutes of Health, Bethesda, MD, U.S.A.

(Received 19 April 1985, accepted 24 May 1985)

Peripheral blood lymphocytes are capable of producing antibody in response to antigenic stimulation by a recall antigen, influenza, during culture for 10 days in medium consisting of 1 part Ham's F-12 and 1 part Iscove's modified Dulbecco's medium supplemented with sodium bicarbonate, bovine crystalline insulin, human transferrin, 2-mercaptoethanol, progesterone, and bovine serum albumin. Anti-influenza antibody levels in the supernatants were determined by enzyme-linked immunosorbent assay. Optimal conditions for production of anti-influenza antibody in this serum-free medium were: influenza concentration, 0.032-0.125 HAU/ml; day of harvest, 10; and cell concentration, 3.0-4.0 x 105 ceils in 200 ~1 of medium per well. Use of serum-free medium will allow examination of the effects of various additives to tissue culture without concern for unknown factors or potential interaction with serum. Key words: human lymphocyte- serum-free- secondary response - influenza - culture medium

Introduction We have previously reported that h u m a n lymphocytes cultured u n d e r serum-free c o n d i t i o n s are capable of surviving, u n d e r g o i n g blast t r a n s f o r m a t i o n a n d biosynthesizing i m m u n o g l o b u l i n ( N e e d l e m a n a n d Weiler, 1981; Sharath et al., 1984). These studies were initiated because we needed a system in which additives could be e x a m i n e d for their effects o n lymphocyte responses without interference from serum in the tissue culture m e d i u m ( N e e d l e m a n et al., 1981; Weiler el al., 1981, 1982; M o r g a n et al., 1982). U n t i l recently, secondary i m m u n e responses in a h u m a n system had not been reported; however, these responses are now being studied c o m m o n l y (Lane et al.,

1 Supported in part by the Veterans Administration and the Kroc Foundation. 2 Recipient of a Clinical Investigator Career Award from the Veterans Administration. 3 Correspondence to: John M. Weiler, SW34E University Hospital, University of Iowa, Iowa City, IA 52242, U.S.A. 0022-1759/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

334

1981; Yarchoan et al., 1981); but not under serum-free conditions. In the present study, we have examined whether'a primed antibody response by human lymphocytes can occur in the absence of serum supplementation. We describe the elicitation and measurement of a serum-free secondary antibody response to influenza virus and the optimal conditions for such a response.

Materials and Methods

Medium preparation The tissue culture medium used in this study was a modification of the medium described by Mosier (1981) and used in our laboratory for pokeweed mitogen-induced immunoglobulin biosynthesis studies (Sharath et al., 1984). Briefly, this medium consisted of 1 part Iscove's modified Dulbecco's medium (Grand Island Biological Company, Grand Island, NY, cat. no. 430-2200), 1 part nutrient mixture F-12 (GIBCO, cat. no. 430-1700) and the following additives: sodium bicarbonate (2 g/l), bovine crystalline insulin (Sigma Chemical Company, St. Louis, MO, cat. no. 1-5500, 5 mg/l), human transferrin (Sigma, cat. no. T-2252, 5 mg/1), progesterone (Sigma, cat. no. P-0130, 6 ~tg/1), 2-mercaptoethanol (Sigma, cat, no. M-6250, 3.5 /~1/1) and gentamicin (Schering Company, Kenilworth, N J, 50 mg/l). Henceforth, this medium is referred to as tissue culture medium (TCM). Virus

The virus used in these studies was formalin-inactivated influenza (strain A/Aichi (A2/Aichi/2/68 MN25241[H3N2])) which was grown in embryonated eggs, zonally purified and provided as a generous gift by Dr. Brian Murphy of the National Institute of Allergy and Infectious Diseases, Bethesda, MD. It was extensively dialyzed against phosphate-buffered saline, pH 7.2 and passed through a 0.22 ~m filter (Coming, Corning, NY). The virus was then quantitated using the standard viral hemagglutinin assay (Hierholzer et al., 1969).

Lymphocyte preparation Peripheral blood was drawn from healthy donors into ethylenediamine-tetraacetate containing vacuum tubes. The mononuclear cells were then isolated by centrifugation through 59% Percoll (Pharmacia Fine Chemicals, Piscataway, N J) as previously described (Sharath et al., 1984). Cell concentrations were determined using a particle counter (Model ZF, Coulter Electronics, Hialea, FL) and cell viability was determined using the criteria of exclusion of 36 mg% trypan blue dye (Phillips, 1973). Tissue culture conditions All culturing was performed using 96-well flat-bottom tissue culture plates (Costar, Cambridge, MA, cat. no. 3596). Each of 6 replicate wells received a total volume of 0.2 ml which contained the appropriate dilution of cells and virus in TCM. The plates were incubated in a 37°C, 5% CO 2, humidified atmosphere. Unless

335 indicated otherwise, the cells were cultured in TCM containing 0.4% bovine serum albumin (Flow Laboratories, McClean, VA, cat. no. 29-101-54) (BSA-TCM) at 3.5 x 105 cells/well (1.75 x 106 cells/ml) for 10 days. Groups of 6 replicate wells were then pooled and centrifuged at 800 × g for 5 min and the supernatants examined for immunoglobulin.

Enzyme-linked immunosorben t assay (ELI S A ) The supernatants were assayed for specific anti-A/Aichi IgG using a variation of the ELISA technique originally described by Engvall and Perlman (1972). We used Immulon II flat-bottom plates (Dynatech Laboratories, Alexandria, VA, cat. no. 011-010-3450); all incubations were performed at 37°C in a 5% CO 2 humidified atmosphere. 150 gl of Tris-HC1 buffer (0.05 M Tris hydroxymethyl aminomethaneHC1, pH 9.5, 0.02% NAN3) containing 1.5 hemagglutinin antigen units (HAU) of virus were added to each well. After a 2 h incubation, each well was washed 5 times with saline-Tween 20 (wash buffer, 0.15 M NaCI, 0.05% polyoxyethylene sorbitan monolaurate) using a semi-automatic washer (Skatron, Sterling, VA, cat. no. 7112). Immediately following the wash, 100 ~tl of tissue culture supernatant or reference serum diluted in PBS-Tween 20 (reaction buffer, 0.01 M NaPO 4, pH 7.2, 0.015 M NaC1, 0.05% polyoxyethylene sorbitan monolaurate, 0.02% NAN3) was added to each well; duplicate serial 2-fold dilutions were run starting at a 1 : 5 dilution for the supernatant and a 1:1000 dilution for the reference serum. The amount of antiA/Aichi in the reference serum was estimated by a method similar to that described by Stevens (1980), as described previously (Yarchoan et al., 1981). After a 2 h incubation, each well was washed with wash buffer and then 100 gl of PBS-Tween 20 containing the appropriate dilution of rabbit anti-human IgG (Cappel Laboratories, Malvern, PA, cat. no. 21013) was added. Again the plates were incubated for 2 h, washed with wash buffer and 100 gl of alkaline phosphatase-conjugated goat anti-rabbit IgG (Cappel Laboratories, cat. no. 21347) diluted in PBS-Tween 20 was added to each well. The plates were allowed to sit overnight in the incubator (15 h), following which each well was washed with wash buffer and immediately filled with 100 gl of Sigma 104 phosphatase substrate diluted to 1 mg/ml in carbonate buffer (0.05 M K2CO3, 0.001 MgCI 2, pH 9.8). The plates were allowed to sit in the incubator until the lowest dilution of the reference serum developed an absorbance of approximately 1.0 at 405 nm. Absorbance was determined using a microELISA autoreader (Model no. MR580, Dynatech Laboratories) or a Bio-Tek Model 310 ELISA reader (Bio-Tek Instruments, Burlington, VT). At this time, the reaction was terminated by the addition of 50 /~1 of 0.3 N NaOH to each well. Specific anti-A/Aichi IgG in tissue culture supernatants was determined by comparison with a reference serum of known titer using the ELISA-CALC computing program (Comple-Software, Iowa City, IA). Results Preliminary studies indicated that donors varied greatly in ability to produce specific antibody to influenza in serum-free medium. Initially we examined 8 donors

336 TABLE 1 COMPARISON OF ABILITY OF PBL FROM VARIOUS DONORS TO PRODUCE ANTI-INFLUENZA A N T I B O D Y Peripheral blood cells from each of 8 donors were cultured in TCM supplemented with 0.4% BSA as described and stimulated with concentrations of A/Aichi influenza virus as shown. Day 10 supernatants were harvested and assayed for IgG anti-influenza virus antibody. Donor

IgG produced ( n g / m l ) HAU/ml 0.5

1

0

2 3 4 5 6 7 8

2 0 8 0 28 0 0

0.125

0.032

19 38 0 100 0 200 0 0

15 0 10 20 0 15 6 0

0.0075

0

0

0

0 0 0 0 20 2 0

0 0 0 0 0 0 0

for IgG biosynthesis using 5 different doses of the virus ranging from 0.0075 to 0.5 HAU/well (Table I). The level of IgG found in the tissue culture supernatants ranged from none to 200 ng/ml with considerable variation among the donors; 2 produced no detectable antibody, and the 6 who responded produced maximal responses ranging from 6 to 200 ng/ml. The maximal response occurred at virus concentrations of 0.032 to 0.125 HAU/ml. It has previously been shown that the ELISA for anti-influenza virus antibody is not significantly affected by virus concentrations up to 0.5 H A U / m l (Yarchoan et al., 1981).

100-

~.~

I0

o

1

I

0,5

l

I

I.~5

2

b

2.~5

3

C E L L S / m l X 10 - 6 Fig. 1. PBL were cultured for 10 days at various concentrations in the presence of influenza virus in TCM with 0.4% BSA. The supernatants were harvested and anti-influenza antibody determined by EL|SA.

337 100-

10

o

0

0 .'5

1'

J 1.5

2

BSA (%) Fig. 2. PBL were cultured for 10 days at 1.75 × 106 ceUs/ml in the presence of influenza virus in T C M with various concentrations of BSA. The supernatants were harvested and anti-influenza antibody determined by ELISA.

Next we examined the conditions necessary to optimize the response in donors who were responsive to the antigen. We varied the day of cell harvest, the cell concentration and the amount of BSA present in the medium. We first established cultures that were harvested on days 1 through 10 and assayed all the supernatants on day 10 for antibody. Maximal antibody production occurred on days 6-9 and total Ig production reached a plateau by days 9-10 (data not shown). Fig. 1 demonstrates that the optimal cell number was between 3.0-4.0 x 10 5 cells/well (1.5-2.0 × 10 6 cells/ml). Fig. 2 shows that medium containing 0.4-0.8% BSA produced optimal amounts of IgG. Finally, we compared the ability of influenza virus to stimulate antibody produc-

T A B L E II C O M P A R I S O N OF 0.4% BSA A N D 10% FCS S U P P L E M E N T E D M E D I A TO S U P P O R T ANTIBODY BIOSYNTHESIS

Peripheral blood cells from each of 3 donors were cultured with 0, 0.1, or 0.4 H A U / m l of A/Aichi influenza virus in TCM supplemented with 0.4% BSA or 10% FCS, and day 10 supernatants were assayed for anti-influenza virus antibody as described. Donor

1 2 3

IgG produced (ng/ml) 0.4% BSA

10% FCS

HAU/ml

HAU/ml

0.5

0.1

None

0.5

0.1

None

400 20 0

120 35 0

3 0 0

> 500 25 0

400 90 3

0 0 0

338

tion in our serum-free medium and in a standard fetal calf serum containing medium (TCM supplemented with 10% FCS). As is shown in Table II, the medium containing 10% FCS produced higher levels of anti-influenza antibody; the serum-free medium in this experiment, however, did allow up to 400 ng/ml of specific IgG to be produced.

Discussion The experiments reported in this paper indicate that human PBL are capable of producing a secondary immune response in vitro using serum-free medium. There is considerable variation in the ability of donor lymphocytes to produce this response with about 75% of the donors producing detectable antibody under optimal conditions. There was an absolute requirement for BSA to be present in the medium for the responses to occur, and optimal responses were found at concentrations of 0.4 and 0.8%. Optimal cell number was 3.0-4.0 x l0 s cells in.200 /~1 per well. The optimal concentrations of influenza virus for eliciting a specific antibody response in this medium (mean concentration = 0.125 H A U/ m l ) was usually slightly lower than that described for a FCS-containing medium (0.5 H A U / m l ) (Yarchoan et al., 1981). The reason for this difference is not clear. We have, however, also previously observed that less antigen is required to stimulate proliferative responses optimally in serum-free medium than in serum-containing medium (Needleman and Weiler, 1981). The experiments described in this paper demonstrate that a serum-free system using chemically defined medium is capable of supporting human PBL to produce secondary immune responses in vitro. This system will allow the study of the effect of additives on the immune reponse without interference and uncertainty caused by the known and unknown substances contained in serum. In addition, these results demonstrate that serum is not necessary for human PBL to produce secondary responses in tissue Culture.

Acknowledgements The authors would like to thank Dr. Thomas Feldbush and Charles Severson for suggestions early in the conduct of these studies and Dr. David L. Nelson for his helpful advice. The authors would like to express their thanks to Ms. Darla Bartels and Ms. Robin Heffron for their excellent secretarial assistance and Luke Sloan for his excellent technical assistance.

References Engvall, E. and P. Perlman, 1972, J. Immunol. 109, 129. Hierholzer, J.C., M.T. Suggs and E.C. Hall, 1969, Appl. Microbiol. 18, 824.

339 Lane, H.C., D.J. Volkman, G. Whalen and A.S. Fauci, 1981, J. Exp. Med. 154, 1043. Misiti, J. and T.A. Waldmann, 1981, J. Exp. Med. 154, 1069. Morgan, E.L., W.O. Weigle and T.E. Hugli, 1982, J. Exp. Med. 155, 1412. Mosier, D.E., 1981, J. Immunol. 127, 1490. Needleman, B.W. and J.M. Weiler, 1981, J. Immunol. Methods 44, 3. Needleman, B.W., J.M. Weiler and T.L. Feldbush, 1981, J. Immunol. 126, 1586. Phillips, H.J., 1973, in: Tissue Culture: Methods and Applications, eds. P.F. Kruse and M.K. Patterson, Jr. (Academic Press, New York) p. 406. Sharath, M.D., S.B. Rinderknecht and J.M. Weiler, 1984, J. Lab. Clin. Med. 103, 739, 748. Stevens, R.H., 1980, in: Manual of Clinical Immunology, eds. N.R. Rose and H, Friedman (American Society for Microbiology, Washington, DC) p. 157. Weiler, J.M., Z.K. Ballas, B.W. Needleman, M.V. Hobbs and T.L. Feldbush, 1982, Immunol. Today 3, 238. Weiler, J.M., Z.K. Ballas, T.L. Feldbush and B.W. Needleman, 1982, Immunology 45, 247. Yarchoan, R., B.R. Murphy, W. Strober, H.S. Schneider and D.L. Nelson, 1981, J. Immunol. 127, 2588.