Eur. J. Immunol. 1979.9: 751-757

Virus infection of lymphoblasts alters the binding affinity of anti-H-2

3 Andrt, C, and Vaerman, J. P., in Hemmings, W. A. (Ed.), Antigen Absorption by the Gut, MTP Press, Lancaster 1978, p. 73. 4 Mattingly, J. A. and Waksman, B. H., J . Immunol. 1978. 121: 1878. 5 Desaymard, C. and Feldmann, M., Eur. J . Immunol. 1975.5: 537. 6 Marbrook, J., Lancet 1967. ii: 1279. 7 Cunningham, A. J. and Szenberg, A,, Immunology 1967.14: 599. 8 Lemaitre-Coelho, I., Andrt, C. and Vaerman, J. P., Prot. Biol. Fluids 1977. 25: 891. 9 Cambiaso, C. L., Goffinet, A,, Vaerman, J. P. and Heremans, J. F., Immunochemistry 1975. 12: 273. 10 McConahey, P. S. and Dixon, F. S., Int. Arch. Allergy Appl. Immunol. 1966. 29: 185. 11 Rittenberg, M.B. and Pratt, K. L., Proc. SOC. Exp. Biol. Med. 1969. 132: 575.

Paul A. Liberti+, Charles J. Hackett’ and Brigitte A. Askonas Division of Immunology, National Institute for Medical Research, Mill Hill, London

751

12 Ey, P. L., Prowse, S. J. and Jenkin, C. R., Immunochemistry 1978. 15: 429. 13 Schreier, M. H. and Nordin, A. A., in Loor, F. and Roelants, G. E. (Eds.), B and T Cells in Immune Recognition, J. Wiley and Sons, London, New York, Sydney, Toronto 1977, p. 127. 14 Uhr, J. W. and Moller, G., Adv. Immunol. 1968. 8: 81. 15 Lees, R. K. and Sinclair, N. R. StC., Immunology 1973. 24: 735. 16 Abraham, S . , Phillips, R. S. and Miller, R. A., J . Exp. Med. 1973. 137: 870. 17 Kappler, J. W., Van der Hoven, A., Dharmarjan, V. and Hoffman, M. K., J. Immunol. 1973. I l l : 1228. 18 Hoffmann, M. K., Kappler, J. W., Hirst, J. A. and Oettgen, H. F., Eur. J . Immunol. 1974. 4: 282. 19 Hoffman, M. K. and Kappler, J. W., Nature 1978. 272: 64. 20 Gordon, J. and Murgita, R. A., Cell. Immunol. 1975. 15: 392.

Influenza virus infection of mouse lymphoblasts alters the binding affinity of anti-H-2 antibody: requirement for viral neuraminidase The requirement for histocompatibility in the T lymphocyte killing of virus-infected cells has led us to investigate the effect of influenza virus infection on mouse cell surface histocompatibility (H-2) antigens. Monoclonal anti-H-2 antibody made it possible to develop equilibrium binding conditions for the assay of H-2 antigen-antibody interactions on intact cells. Scatchard analysis of anti-H-2 binding with normal and virus-infected cells yielded linear curves indicating homogeneity of the interaction at varied concentrations of antibody through saturating levels. T h e estimated number of 2 x lo5 - 5 x lo5 H-2 antigenic sites per mouse lymphoblast does not appear to change during the course of influenza virus infection. However, the K, (binding affinity constant) of anti-H-2 binding is rapidly elevated by virus infection (“0” time), continues to increase for 3 h post infection, then decreases. Control cells, treated with normal egg allantoic fluid, show n o change in K, during similar incubation. This change in K, requires the presence of active viral neuraminidase. Thermal denaturation of the neuraminidase of the virus particles abolishes their ability to induce K, alteration, even though hemagglutinin activity is retained. Treatment of cells with neuraminidase of bacterial origin led to an elevation of K,, but did not mimic the viral effect in time dependence and magnitude of peak responses. The time-dependent lowering of K, from peak values appeared to relate to virus replication, since UV light-inactivated virus-induced K, elevation, but did not produce the typical K, decline at 4-5 h post infection. T h e changes in K, of anti-H-2 binding during influenza infection reflects a virusinduced alteration of the H-2 molecule o r its environment in the host cell membrane. The molecular basis of this change and its relation to H-Zrestricted recognition of influenza virus-infected cells by cytotoxic T cells requires further study.

[I 24251 +

Recipient of a Public Health Research Career Development Award from NIAID, NIH. Present address: Department of Biochemistry, Jefferson Medical College, Philadelphia, PA 19107, USA. Fellow in Cancer Research supported by Grant DRG-207-F of the Damon Runyon-Walter Winchell Cancer Fund.

Correspondence: Brigitte A. Askonas, Division of Immunology, National Institute for Medical Research, Mill Hill, London NW 7 1AA. GB

Abbreviations: BSA: Bovine serum albumin HA: Hemagglutinin LPS: E. coli lipopolysaccharide N: Neuraminidase PBS: Phosphate-buffered saline 0 Verlag Chemie, GmbH, D-6940 Weinheim, 1979

1 Introduction The specific immune killing of virally infected cells [l-31, tumor cells [4], or chemically modified cells [5] by cytotoxic T cells is restricted by histocompatibility antigens (K or D end of H-2 in mouse [3], or HLA in man [6]). This implies that both virus-specific proteins and histocompatibility antigens need to be recognized by the T cells. Two models have been proposed to account for this recognition requirement, either dual T cell receptors recognizing virus and H-2 separately, o r a single receptor recognizing “altered self” which could result from the interaction of virus and H-2 [3]. As the nature of the T cell receptors remains obscure, we wished to find out whether virus-cell interactions lead to any detectable changes in H-2. 0014-2980/79/1010-0751$02.50/0

752

P. A. Liberti, C. J. Hackett and B . A. Askonas

The selection of hybrid clones forming monoclonal antibodies to H-2Kk by Lemke et al. [7] makes it possible to develop conditions for equilibrium binding of anti-H-2 to cells and to obtain measurements of affinity constants for antibody H-2 interactions. We chose cytotoxic T cell killing of influenza virus-infected cells [8] as a model system for this type of study. The target cell killing is restricted by the H-2 locus, and since virus-infected lymphoblasts serve as good target cells [8-101, any strain combination can be tested. Influenza virus infection of tumor cells or lymphoblasts is abortive, but viral antigens are produced and expressed on the host cell surface [ l l , 121. We utilized monoclonal antibodies to look for changes in H-2 on the cell surface during the course of influenza virus infection. Using Scatchard analysis in a manner entirely analogous to systems employing homogeneous ligand interacting with antibodies of multiclonal origin [13, 141, we found that although the number of H-2 antigenic sites remains constant, there is a significant time-dependent alteration of the H-2 anti-H-2 binding affinity constant following influenza virus infection. To induce this alteration, the virus must possess active neuraminidase.

2 Materials and methods 2.1 Cells

The preparation of spleen lymphocyte suspensions has been described [ E l . Lymphoblasts were prepared from normal CBA (H-2k)mice by incubating lo6 spleen cells/ml for 3 days in RPMI 1640 culture medium plus 10% (vlv) heat-inactivated fetal calf serum (RPMI/10) in the presence of 5 x 1 0 p 5 2~ mercaptoethanol and Escherichia coli lipopolysaccharide (LPS) at a 10 pg/ml concentration [9]. Prior to use, large blast cells were purified on bovine serum albumin (BSA) gradients ~51. 2.2 Monoclonal antibodies

Two monoclonal antisera to H-2Kk, produced by hybrid cell lines 3 0 R 3 (IgG2, specificity 5 ) and 2 7 R 9 (IgG2b specificity 25), were the kind gift of Dr. H. Lemke, Institute of Genetics, Cologne [7]. Monoclonal antibodies were partially purified on a staphylococcal protein A-Sepharose column by stepwise p H elution [16]. The protein A column (0.75 ml bed volume) was equilibrated with 0.05 M Tris, 0.1 M NaC1, pH 8.5. Typically, 100-200 pl of antiserum, diluted with an equal volume of the Tris-NaC1 buffer, was applied and the column washed with 10 bed volumes of the same buffer, followed by 10 bed volumes of 0.05 M Citrate, 0.1 M NaCI, pH 6.0. IgG2, and IgGZb were eluted with 0.05 M glycine, 0.1 M NaC1, p H 3.0. Fractions (0.5 ml) were neutralized immediately with 70 pl of 1 M Tris, pH 8.0. Total protein recovered in the glycine buffer eluate (determined by A280)ranged from 180-400 pg. Eluted Ig was then dialyzed against the appropriate buffer for 1251 labeling by either the chloramine-T method [I71 or the Bolton-Hunter reagent [MI. Radiolabeled protein (separated from the iodination reagent by gel filtration) was made 1.75% in BSA, dialyzed overnight against binding buffer (RPMI 1640 containing 1.75% BSA, 0.2% NaN3, pH 7.0) and centrifuged at 23000 X g for 30 min before use.

Eur. J. Immunol. 1979.9: 751-757 Specific activity of radiolabeled antibody preparations ranged from 2.3 X lo4 to 2.8 x lo5 c p d p g protein. Radioactivity in the samples attributable to active antibody was determined by exhaustive serial adsorption of labeled antibody onto CBA spleen cells: 100 pl of the preparation was incubated with lo6 cells for 90 rnin with agitation at 15-min intervals. After centrifugation, the supernatant was added to a further lo6 cells, and this process was repeated twice more. About 24 to 28% of the labeled Ig represented antibody. For each iodinated preparation, H-2 specificity was checked using BALB/c (H-2d) spleen cells or lymphoblasts. Nonspecific binding was negligible (see Sect. 3.1). 2.3 Antibody-binding studies

Spleen cells or lymphoblasts exposed to various treatments were harvested at desired time intervals, centrifuged, and resuspended at 5 x 106-2 x lo7 celldm1 in binding buffer (RPMI 1640 containing 0.05 M HEPES, 1.75% BSA, 0.2% NaN3, pH 7.0). Aliquots (100 pl) of the suspended cells were added to plastic tubes (Milian Instruments) containing 30 p1 '251-labeled antibody diluted to different concentrations with binding buffer. The suspension was gently stirred in a Vortex mixer at 15-min intervals for 90 min, by which time binding equilibrium had been achieved. Unbound material was removed by diluting the reaction mixture with 400 p1 binding buffer, followed by centrifugation. The cell pellets were washed twice more and their radioactivity determined in an LKB Wallac gamma counter. All operations were performed at room temperature (see Sect. 3.1). 2.4 Influenza viruses

Type A influenza virus strains X-31(H3N2) = A/Hong Kong/l/ 68 X A/PR/8/34, and JapIBel (H2N1) = A/JAPAN/305/57 X A/ BEL/42 were used. Viruses were cultivated in embryonated chicken eggs, and allantoic fluid was titrated for hemagglutinin (HA) activity. The virus preparations were gifts of Dr. J. J. Skehel. Inactivation of virus was carried out by UV light treatment (2 min exposure, 20 cm distance from a Philips 25 W germicidal bulb). 2.5 Neuraminidase (N) experiments Viral N can be inactivated while H A activity is preserved. To establish N inactivation kinetics, 1 ml aliquots of allantoic fluid (AIJAPIBEL) were heated to 45 or 50°C in a water bath. Samples removed at various time intervals were incubated at 37°C with fetuin (50 mg/ml) in phosphate-buffered saline (PBS) made lop4M in CaC12. Released sialic acid was assayed by the thiobarbituric acid method of Warren [19]. Optimum N inactivation was found to require 60 min at 55°C. Prior to H A titration, such heat-treated virus suspensions were ultrasonicated to disperse aggregates. Virus particle-mediated release of sialic acid from cells was determined by adding an infective dose of virus to lymphoblasts in PBS M CaC12 and incubating at 37°C for 60 min. Sialic acid in the supernatant was then determined. N treatment of viable cells was at the level of 100 units of Vibrio cholerae N (Behringwerke, MarburgILahn, FRG) in 0.2

Eur. J. Immunol. 1979.9: 751-757

Virus infection of lymphoblasts alters the binding affinity of anti-H-2

753

ml isotonic PBS, lop4 M CaC12, 60 min at 37°C. Cells were pelleted and supernatants assayed for sialic acid. Cell viability was not affected by this enzyme treatment.

biphasic curve would indicate IgG binding to two H-2 antigens at low antibody concentrations, but switching to a 1 : 1 ratio of antibody to H-2 in antibody excess.

Total releasable sialic acid was estimated by mild acid hydrolysis of 3 x 10' - 10 xio7 cells in 0.1 N H2S04in isotonic saline, M CaC12, 1 h at 80°C, followed by centrifugation and determination of sialic acid in the supernatant.

Fig. 1 illustrates the Scatchard plot of 27 R 9 antibody binding to CBA spleen cells in the absence of azide, and the biphasic curve strongly suggests that the ratio of antibodyiantigen binding changes to 1: 1 with increasing antibody concentrations. However, in the presence of 0.2% azide, the plot is linear over the entire concentration range examined indicating antibody/ antigen binding at a ratio of 1: 1. Identical K, values of 1.6 X 10' 1 / can ~ be calculated from the linear parts of both curves. Azide was therefore always included in the binding assays, but was not present in prior treatment of cells (such as virus infection).

2.6 Infection of cells with influenza virus

Spleen cells or lymphoblasts (lo'lml) in RPMI 1640 medium were infected with 800 H A units of influenza virus, in allantoic fluid, or purified as indicated. After 1-2 h infection at 37"C, the cells were washed and incubated for another 3 4 h at 37". Control cells were treated similarly, but with normal allantoic fluid. UV light or N-treated virus was titrated for H A activity, and the same number of H A units were added to the cells.

Extrapolation of the Scatchard curve to the abscissa gives a value of n = 1.75 x105 H-2 siteslspleen cell. With gradientTable 1. Binding characterization") of partially purified antibody to H-2 Kk

3 Results BALBlc spleen lymphocytcs

3.1 Optimal conditions for antibody-binding assays

Preliminary experiments over a range of temperatures and cell concentrations (not illustrated) were performed to establish optimum conditions for the binding of monoclonal anti-H-2 to splenic lymphoid cells. Using 1251-labeledantisera (30 R 3 and 27R9), cell densities of 5 x 106-2 x lo7 cellslml proved optimal. A 90-min incubation, agitating at 15-min intervals, is required to reach equilibrium binding. The presence of BSA (1.75%) reduces nonspecific adsorption to background levels, and 0.2% azide markedly improves reproducibility of binding and prevents capping. The 27 R and 30 R antisera were sufficiently high in monoclonal antibody to achieve saturation binding. Since curves from binding studies done at room temperature (21 "C) were very reproducible, all further binding was measured at this temperature.

CBA spleen lymphocytcs

CPm added

Igh' cpm bound

bound

cpm added

2624 4410 7500 9460 11240 18444 24 170

34 136 39 I(J1 103 109 152

1.3 3.1 0.5 1.1 0.9 0.6 0.6

3 032 47W 7608 I I 00.1 12760 19620 24866

?h

Igh' cpin hound 540 770

1194 1706 181 1 2732 3164

$4,

bound 17.X 16.4 15.7

15.5 14.2 l3.Y 12.7

a) Binding of 30 R 3 antibody to spleen cells as described in Sect. 2.3. b) Purified Ig fraction iodinated by the cloramine-T method, yielding 2.8 x lo5 cpmlpg.

3.2 Equilibrium binding characteristics

Table 1 shows that if increasing amounts of radiolabeled, partially purified anti-H-2 Kk fractions are incubated under equilibrium conditions with BALBic or CBA spleen cells, radioactivity associates specifically only with the CBA cells. The decrease in % binding with increasing amounts of antibodies is characteristic of multivalent ligand binding, as can be shown by the Scatchard equation [20, 211 in the following form: [anti-H-2bo,,d] [anti-H-2fr,,] x [cell]

= nK,-K,

X

[anti-H-2b,,,d] [cell]

were [anti-H-2b,,,d] and [anti-H-zf,,,] are the equilibrium concentrations in mol/l monoclonal anti-H-2, either bound o r free, [cell] is the molar concentration of cells (celldl : Avogadro's number), n is the number of H-2 antigenic siteslcell, and K, is the equilibrium affinity constant. It can be shown that the above equation is rigorously correct when one antibody molecule binds one antigen 1141. - - Hence, plots of [anti-H-2boU,d/ [anti-H-2fr,,] x [cell] vs. [anti-H-2bound]/[ce11] should yield a straight line of negative slope equal to -Ka. In contrast, a I

Figure 1. Scatchard plots of the binding of 27 R 9 monoclonal antibody to CBA spleen lymphocytes. Binding equilibrium was established in the presence (0)or absence (A) of 0.2% azide at room temperature, 21 "C. Cell viability remained constant at 94% during the attainment of binding equilibrium.

754

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P. A. Liberti, C. J. Hackett and B. A. Askonas

purified populations of larger blast cells, the 27 R 9 antibody detected 20% more H-2 molecules/cell (data not shown). Similar values ranging from 2 x lo5 - 5 x lo5 sites/lymphoblast were obtained with 30 R 3 antibody in experiments reported in the following sections. It is noted that the number of H-2 sites/ cell mist be considered an estimate, since at [anti-H-2,,,,d]/ [anti-H-2kee]X [cell] = 0, the value n = [anti-H-2b,,,d]/[ce11] is extremely sensitive to slight errors in cell counts between various curves. On the other hand, K, values are accurate between curves in a given experiment, since their determinations are mathematically independent of cell concentrations (i.e. [cell] is in the denominator on both axes). Hence, K, values should be in error only to the extent that antibody concentrations (spec. act.) are inaccurate; but since only a single lz5I-1abeledmonoclonal preparation was used for a given experiment, relative distinctions in K, are accurate.

3.3 Effect of virus infection on K, of anti-H-2 binding

The effect of virus infection on H-2 disposition of lyrnphoblasts was studied by infecting 3-day LPS-stimulated CBA lymphoblasts with type A/X-31 influenza virus. Fig. 2 shows the Scatchard plots of antibody binding data using 30 R 3 antibody at various time intervals after virus infection. Control cells treated with virus-free allantoic fluid exhibit similar slopes at all intervals through 5 h of incubation, with K, ranging from 7 x 10’ - 9 x 10’. However, for virus-infected cells (Fig. 2), the slopes of the antibody-binding curves indicate a time-dependent elevation in the binding K,; at 2 h (i.e. at the completion of the virus adsorption) K, was 1.3 X lo9; it reached a maximum of 2.5 x lo9 at 3 h post infection and then declined to 1.8 x lo9at 4 h and 1.3 x lo9 by 5 h. The viability of infected lymphoblasts remained nearly constant (88-92%) over the entire time course. The number of anti-H-2-binding sites in control and infected cells ranged from 2 X 10’ -2.5 x 105/cell.Infection with A/Jap/Bel yields similar results, while lymphoblasts treated with normal alantoic fluid have essentially identical K, (range 1.3 x 10’ - 3.1 X10’) for all incubation times sampled. This K, elevation is evident immediately after interaction of virus (NJaplBel) with the lymphoblasts (“0” time, K, of 1.5 x lo9). The K, peaked at 2 h of infection to 2.5 x lo9, then declined to 1.3 x lo9 and 5.1 X 10’ in the 4 and 5-h samples, respectively (Fig. 3). In this experiments, the maximum alteration in K, reflects an order of magnitude change, i. e. a 1.4 Kcal increase in binding energy. Qualitatively identical results were obtained using the 27 R 9 antiserum, including the rapid K, elevation at “0” time (data not shown). This indicates that such effects are not restricted to a particular H-2 Kkspecificity.

Figure 2. Scatchard plots of the binding of purified 30 R 3 antibody to normal and A/X 31 virus-infected cells. CBA lymphoblasts were infected with A/X31 virus (800 HA units/lO’ cells) or controls were treated with an equivalent volume of normal egg allantoic fluid. After 2 h of virus adsorption, cells were washed and cultured for an additional 3 h. At intervals, binding of purified ‘2sI-labeled30 R 3 Ig was assayed. Affinity constants, determined from the slopes of the resulting Scatchard plots were: Control cells: 2 h (0)8.8 X 10’; 3 h (0) 9.0 x 10’; 4 h (A) 7.0 X 10’; 5 h (V) 8.0 x 10’. Infected cells: 2 h (0) 1.3 X 10’; 3 h (M) 1.6 X lo9; 4 h (A) 1.7 X 10’; 5 h (V) 1.4 x 10’. The moles of cells in each sample were normalized to the 5-h sample to compensate for cell counting errors; slopes, i.e. K, are not altered by this procedure.

-9

15-

I

P 10-

0 2 3 5

0 2 3 5

Incubation tima

p c q Control-. 3.4 Requirement for viral N activity in producing K, elevation In view of the rapidity of the virus effect on K, (i.e. “0” time), and since influenza virus is known to have a N component, we explored the possible role of the active viral enzyme on the increase in K, of anto-H-2 binding. As shown in Table 2, intact virus particles release sialic acid efficiently from cell surface glycoproteins, (ie. 43% of total sialic acid is released).

(h)

I

With A/Ja+el

Figure 3. Increase in K, of anti-H-2binding after infection with A/Jap/ Be1 influenza virus. CBA lymphoblasts were treated either with normal allantoic fluid or infection with A/Jap/Belvirus (800 HA units/107 cells). After 1 h of incubation with virus, cells were washed and incubated for up to 4 h. At intervals, binding of purified ’251-labeled30 R 3 Ig was assayed. The “0” time analysis was done on cells that had been diluted with 5 vol. buffer (plus azide) immediately after mixing with virus or normal allantoic fluid.

Virus infection of lymphoblastsalters the binding affinity of anti-H-2

Eur. J. Immunol. 1979.9: 751-757

Table 2. Sialic acid released from lymphoblasts by various treatments Treatment

p mol sialic acid released/ceII"'

% of total

5.12 x 10-'" 2.22 x 10-10 1.63 X lo-''

100 43 32

Acid hydrolysisb) N JAP/BEL') Commercial N")

a) Sialic acid assayed by the thiobarbituric acid method of Warren ~91. b) Hydrolysis in 0.1 N H2S04 in isotonic saline, 8 0 T , 1 h. c) Virus at 800 HA units/107 cells, 37"C, 1 h., in PBS + CaCI2. d) 100 units of N in PBS + CaCI,, 37°C 1 h.

l

45;

755

Despite the fact that so much more allantoic fluid was added, virus possessing denatured N is virtually without effect on K, of H-2-anti-H-2 interaction; these virus particles can attach to the cells due to their H A activity. Even after several hours' incubation, the K, remained in the range of 2.9 x 10' 3.3 x lo8 (Table 3), in close agreement with control cell values (see Fig. 3). 3.5 Effect of N treatment of lymphoblasts on K, of anti-H-2 binding Since the changes in K, required an active viral N we treated cells with commercial N to test whether changes in binding of anti-H-2 could solely be ascribed to action of the viral enzyme. Antibody binding to cells treated for 1 h with commercial N (which released 32% of available sialic acid residues) showed higher K, values at various time intervals, as compared to the control cells (Table 4). However, the K, of treated cells did not show the time-dependent rise and subsequent decline, as seen during the sequence of virus infection; nor were the dramatic K, peak values, as seen in Fig. 3, obtainable by enzyme treatment alone.

\

0

Table 4. Binding of anti-H-2 to N-treated lymphoblastsa)

\

251

N treatment

Time of incubation (h)

K,

Exposure time (MIN)

Figure 4. Thermal inactivation of A/Jap/Bel N activity. Allantoic fluid (1 rnl) containing type A/Jap/Bel virus was treated in a water bath at 45°C (0)or 55°C (0).Released sialic acid was assayed in lOO-pl samples according to Warren [19].

Table 3. K, of anti-H-2 binding to lymphoblasts treated with denatured N type A/JAP/BEL virusa)

K.

Time of sampling

"0" time 2h 3h 5h

3.2 3.3 2.9 3.1

x Id x 10s x 10" x l@

a) lo7 CBA lyrnphoblasts were treated with 800 HA units of denatured N type NJAP/BEL virus (thermally inactivated as in Fig. 4). To compensate for loss in HA activity, 6 X the usual volume of allantoic fluid was required to give 800 units. K, values were derived from Scatchard analysis of the binding data using purified '251-labeled 30 R 3 antibody; conditions as in Fig. 3.

a) Cells were incubated with 100 units of commercial N for 1 h as specified in Table 2. Controls were cells in PBS plus CaCI2 under parallel conditions. After removal of enzyme, cells were incubated in RPMVlO for various time intervals. K, values were obtained from Scatchard plots of binding of purified 30R3 antibody.

3.6 Effect of inactivated virus on K, of anti-H-2 binding

Treatment of lyrnphoblasts with purified NJaplBel virus either in its native or W-inactivated form elicited elevated K, values, as compared to control cells (Fig. 5). This elevation was evident at "0" time and increased with time of incubation. However, the K, of antibody binding to lymphoblasts treated with inactivated virus failed to decline at 4-5 h, as seen with intact virus. Thus, active viral infection is not a prerequisite for producing the elevated K,, but the K, decline with time appears to be related to virus replication.

4 Discussion N can be inactivated by treating NJaplBel containing allantoic fluid at 55°C for 60 min (Fig. 4). H A activity is reduced, and lymphoblasts were treated with 800 H A units as above (6-fold more allantoic fluid).

Monoclonal antibodies to defined H-2 specificities enabled us to study changes in H-2 antigens on the cell surface during influenza virus infection. The high concentration of antibody

756

P. A. Liberti, C. J. Hackett and B. A. Askonas

i

The evident effect at “0” time indicates that some rapid alteration on the cell surface is occurring after interaction with influenza virus. The viral N turned out to be important for this change. Using A/Jap/Bel virus which retained H A activity after thermally denaturing the N, it was found that the K, values remained at the control level. Thus, the time-dependent elevation of K, values in virus-infected cells requires the presence of intact viral N activity.

L 10

1-

Eur. J. Immunol. 1979.9: 751-757

20 cell

30

lo-’

40

\ \ \~.

Figure 5. Scatchard plots obtained from binding of 30 R 3 antibody to CBA lymphoblasts treated with native or UV-irradiated preparations of purified A/Jap/Bel. CBA lymphocytes were incubated with either native virus or UV-inactivated virus at 800 HA units/107cells. Control cells were incubated with an equivalent amount of PBS. Other conditions are given in the legend to Fig. 3. The affinity constants, determined from these curves are: control cells at “0” time (0)1.5 X lo8; native purified virus-infected cells at “0” time (0)2.4 X 10’; UVirradiated purified virus “0” time (0) 3.2 X 10’ and at 5 h (A) 3.9 x 108.

in the purified IgG fractions and its monospecificity permitted equilibrium binding over a range of antibody concentrations through saturating levels and analysis by the Scatchard equation. Linearity of the Scatchard plots indicated that monoclonal antibodies are homogeneous in their interaction with H-2 sites, as expected. This analysis permits an estimate of the number of H-2 antigenic sites per cell. Values of 2 x lo5 - 5 x lo5 H-2 sites per cell were obtained for large blast cells purified on BSA gradients, using 3 0 R 3 antibody. This serum appears to detect H-2 public specificity 5 [7], which is expressed at both the K and D ends of H-2. Although error in cell concentration introduces inaccuracy into the quantitation of H-2 sites (see Sect. 3.2), our values are very similar to the estimates of 3.75 X lo5 HLA antigens per tonsil cell determined by Barnstable et al. using monoclonal antibody [22], and 5 X 10’ HLA molecules per human lymphocyte from P2-microglobulin assays of Plesner [23] (assuming that virtually all the P,-microglobulin is associated with HLA molecules). We consistently detected fewer H-2 sites on spleen cells as compared to larger lymphoblasts; this is likely to reflect the larger surface area of lymphoblasts. However, we saw no consistent changes in the number of H-2 sites during the course of virus infection. In contrast, it was found that cells infected with native influenza virus have elevated K, values compared to uninfected control cells. This effect was time-dependent, being evident at “0” time (cells mixed with virus, then immediately washed and assayed for binding), reaching a maximum 2-3 h after infection, but declining at 4-5 h. This was observed with two type A influenza viruses, X-31 and A/Jap/Bel and with two monoclonal antibodies of different H-2 Kk specificities [7]. Control cells treated with allantoic fluid bound the antibody with essentially a constant K, throughout the same time course.

The viral N behaved differently from commercial N although both enzymes cleaved similar amounts of sialic acid from the cells. Commercial enzymes led to only minor increases in K,, and no further time-dependent changes took place. Inactivated virus (by UV light) with active N still induced elevation of K, at “0” time and at 3 h, but subsequent decreases in K,, observed with infective virus, did not occur. This would relate the late decrease in affinity of antibody binding to the replication of viral proteins and their expression on the cell surface ([11, 121 and S. Courtneidge, unpublished results). Conceivably, these viral proteins could block or interfere with antibody accessibility to the H-2 molecules, either by direct association or by indirect perturbation. However, it is not possible at this time to rule out causes due to other metabolic events, such as membrane turnover or capping. The increased assocation constant of antibody binding to H-2 would suggest an alteration of pre-existing H-2 molecules after interaction of virus with the cell surface. We conclude this on the basis of the rapid K, alteration early in the infective process and the finding that the number of H-2 sites per cell does not change significantly during virus infection. Therefore, all the H-2 molecules continue to bind the antibody after infection, but the binding affinity is altered. Possible causes could include conformational changes in the H-2 molecules, or an increased accessibility to the antigenic sites. Further studies are needed to determine whether viral neuraminidase acts directly on the H-2 molecule, or whether indirect interactions in the membrane are causal. These results indicate that complex events are occurring on the cell surface during the sequence of influenza virus infection, reflected in alteration of H-2-anti-H-2 association constants. Whether the H-2 molecules is preferentially associated with virus, as was detected by Bubbers and Lilly [24], in virions of Friend virus, or whether the virus in the membrane induces an indirect change in the H-2 molecule, is not yet clear. A recent report by Helenius et al. [25], showing that Semliki Forest virus specifically binds to histocompatibility molecules, may be relevant to our findings. In this context, it should be mentioned that altered expression of H-2 antigenic sites on lymphocytes has been elicited in response to interferon treatment [26, 271 and following in vivo infection with radiation leukemia virus [28]. Although in these cases, the treatments appear to stimulate increased H-2 glycoprotein synthesis rather than the immediate alteration of pre-existing molecules, as occurs with influenza virus infection, the general involvement of the H-2 molecule in virus attachment, interaction with virus particles, and response to interferon treatment may be related to the critical role of histocompatibility molecules in defense against virus infection. Although it is definite that the condition of the histocompatibility molecule is altered rapidly during infection, and that action of viral N is required, further studies are needed to determine the structure or sites which may be important in recognition by cytotoxic T cells.

Eur. J. Immunol. 1979. 9: 757-761

Phospholipase C-induced lysis

The authors are most grateful to H . Lemke and K . Rajewsky for their generous gift of monoclonal antibody. We also wish to thank R. G. Webster, R . M. E. Parkhouse and G. G. B. Klaus for helpful discussions and Dianne Millican for expert technical assistance.

Received February 15, 1979.

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Cytotoxicity of human mononuclear cells against chicken and human red blood cells, induced by treatment of the effector cells with phospholipase C Human mononuclear cells from peripheral blood which were treated with phospholipase C (PLC), became cytotoxic against human or chicken red blood cells. PLCinduced cellular cytotoxicity against human red blood cells was further analyzed and compared to anti-D-mediated, antibody-dependent cellular cytotoxicity (ADCC), using the same target cells. ADCC, but not cytotoxicity of PLC-treated effector cells, was inhibited by free IgG. In addition, iodoacetate strongly enhanced PLC-induced cytotoxicity, but blocked ADCC completely. Addition of fetal calf serum or human AB serum impaired PLC-induced cytotoxicity. A similar inhibition was found by adding lecithin liposomes suggesting that the inhibitory effect of sera was also due to their phospholipid content. The data show that cytotoxicity of PLC-treated effector cells can be clearly distinguished from cellular cytotoxicity, occurring spontaneously or induced by target cell antibodies. We favor the notion that cytotoxicity of PLCtreated effector cells against human erythrocytes is due to the action of PLC, adsorbed to the effector cells.

[I 20491 Correspondence: Klaus Resch, Institut fur Immunologie und Serologie, Im Neuenheimer Feld 305, D-6900 Heidelberg, FRG Abbreviations: PBS: Phosphate-buffered saline HEPES: 2-[h-(Hydroxyethy1)-1 piperazine] ethane sulfonic acid DMEH. Dulecco’s modified Eagle’s medium, buffered with 20 mM HEPES FCS: Fetal calf serum ABS: Human AB serum PLC: Phospholipase C CRBC: Chicken red blood cells HRBC: Human red blood cells ADCC: Antibody-dependent cellular cytotoxicity 0 Verlag Chemie, GmbH, D-6940 Weinheim, 1979

1 Introduction Modification of effector cells by various enzymes has been widely used to elucidate the involvement of surface structures in the mechanism of target cell destruction [l, 21. Recently, lymphoid cells from human peripheral blood have been shown by Saksela et al. [3, 41 to become strongly cytotoxic against chicken red blood cells (CRBC) upon treatment with phospholipase C (PLC). The authors called the phenomenon “augmentation” of spontaneous cytotoxicity. In their experi0014-2980/79/1010-0757$02.50/0