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Influenza virus changes cell-surface glycoproteins including major histocompatibility complex determinants on lymphocytes
Influenza Virus Changes Cell-Surface Glycoproteins Including Major Histocompatibility Complex Determinants on Lymphocytes Francien T.M. Rotteveel, Jacques J. Neefjes, Hidde L. Ploegh, and Cornelius J. Lucas
A B S T R A C T : The effect of influenza virus infection on the expression of major histocompatibility complex (MHC) antigens was investigated. Infection with influenza virus resulted in an increase of the binding of anti-MHC class I and class II antibodies to resting T cells. The binding of anti-MHC class II antibodies to activated T cells was increased approximately threefold. The binding of anti-MHC class I and class II antibodies to Epstein-Barr virus -transformed B cells appeared unaffected after influenza virus infection. Recombinant human interferon-~ and/or -7 added to T cells did not enhance the binding of anti-MHC antibodies. Biochemical analysis revealed no increase in the amount of class I and class II antigens as a consequence of viral infection, but a marked decrease in sialic acid content was found, most probably caused by the viral neuraminidase. Pulse-chase experiments suggest that the viral neuraminidase can catalyze the removal of sialic acids both en route to and at the cell surface. The absence of sialic acid residues can explain the increased binding of anti-MHC antibodies, because neuraminidase (clostridium perfringens) treatment of T and Epstein-Barr virus-transformed B cells resulted in a shift in both isoelectric point and antibody binding similar to that observed after influenza virus infection. ABBREVIATIONS
APC EBV FCS IFN MHC
antigen-presenting cells Epstein-Barr virus fetal calf serum interferon major histocompatibility complex
MoAb 1D-IEF PHA
monoclonal antibody one-dimensional isoelectric focusing phytohemagglutinin
INTRODUCTION
The products encoded by the major histocompatibility complex (MHC) guide lymphocytes in their interactions with antigen-presenting cells (APCs) or target cells. CD4 ÷ cells predominantly utilize M H C class II molecules as restriction From the Central Laboratory of the Netherlands Red Cross Blood Transfusion Serviceand the Laboralory for Experimental and Clinical Immunology, University of Amsterdam (F.T.M.R., CJ.L.) and the Netherlands Cancer Institute, Amsterdam, the Netherlands (J.J.N., H.L.P.). Address reprint requests to Dr. CorneliusJ. Lucas, c/o Publication Secretariat, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, P.O. Box 9406, 1006 AK Amsterdam, the Netherlands. ReceivedJanuary 17~ 1989; revisedMay 8. 1989.
Human Immunology26, 199-213 (1989) © AmericanSocietyfor Histocompatibilityand Immunogenetics,1989
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F.T.M. Rotteveel et al. elements, whereas CD8 ÷ T cells preferentially recognize antigens presented by M H C class I determinants. It has been shown that the level at which M H C products are expressed on APCs is an important parameter in determining the ability to present antigen to T lymphocytes [1-3] and in T-/B-cell interactions [4,5]. The absence of M H C class I antigens renders a cell resistant to attack by cytotoxic CD8 ÷ T cells. Similarly, a cell devoid of class II antigens cannot present a processed foreign antigen to a CD4 + T cell. A dose-response relationship has been established for the amount of M H C class I antigen, and the ability to induce a cytotoxic T lymphocyte response in vitro [6]. A threshold level of M H C class l I antigens is also required to trigger an immune response, the magnitude of which increases concomitantly with class II antigen expression [7]. Thus, the levels of M H C antigen determine whether or not stimulation of responder cells wilt ensue. Several viruses have been shown to modulate the level of MHC determinants in APCs or target cells. Certain viruses abrogate expression of M H C class [ [8-10] and/or class II antigens [11], whereas others cause an increase in levels of M H C class I [6] or class II [12,13] determinants. This control of expression of M H C antigens is exerted at different levels. A decrease in expression may bc caused by transcriptional and/or posttranscriptional processes [8,14], Examples of both types have been documented. An increase in expression may be due to the production of and response to interferons, potent cytokines that generally increase levels of class I [interferon (IFN)-~, -/3, -5~] and class II (IFN-50 on many susceptible cell types [15-19], an effect that may be potentiated by tumor necrosis factor [20]. In the present report, we describe the effect of influenza virus infection on M H C class I and class II molecules. We show that removal of sialic acids by the virally encoded neuraminidase results in increased binding of anti-MHC monoclonal antibodies (MoAbs), the actual levels of MHC-encoded potypeptides being identical in virus-infected and control cells. Analysis of the biosynthesis of M H C products in virus-infected cells suggests that removal of sialic acids takes place both intracellularly and at the cell surface.
MATERIALS AND METHODS Cells Peripheral blood mononuclear cells (PBMCs) were obtained by Ficoll-Hypaque density centrifugation of peripheral blood from healthy donors. T cells were isolated by sheep erythrocyte (E)-rosette sedimentation. Activated cells were prepared by culture of PBMCs in the presence of phytohemagglutinin (PHA) (5 /.*g/ml; Gibco Laboratories, Grand Island, NY) for 3 days. B-lymphoblastoid cell lines were established by infection of B cells with Epstein-Barr virus (EBV). The B-cell lines were grown in RPMI-1640 containing I0% fetal calf serum (FCS). The monocyte population was obtained by countercurrent centrifugal elutriation. Interferon Recombinant human IFN-o~2b and IFN-5/were generously provided by Dr. van der Meide (TNO, Rijswijk, The Netherlands). The anti-IFN antibodies were used at a concentration of about 3000 neutralizing U/ml.
Influenza Changes Cell-Surface Glycoproteins
201
Virus Influenza A / H K [A/Hong Kong/8/68-X-31 (H3N2)] was a generous gift from Prof. dr. N. Masurel (Erasmus University, Rotterdam, The Netherlands). Influenza A/X79 and influenza B/Singapore were gifts from Duphar (Weesp, The Netherlands).
Virus Infection of the Cells Activated T cells (cultured for 2 days with PHA), unstimulated T cells, or B cells were incubated for 90 min with influenza virus. After infection, cells were washed and resuspended in RPMI containing 10% FCS and incubated at 37°C for 18 hr.
Monoclonal Antibodies The MoAb directed against HLA class I determinants, W6/32 (Hb 95), was obtained through the American Type Culture Collection (ATCC, Rockville, MD) [21]. MHC class II-specific MoAbs, R3E2, DA6.231, and IVA12 were used. R3E2 was provided by Dr. M. A. de Rie [Central Laboratory of the Red Cross Blood Transfusion Service (CLB), Amsterdam] and recognizes nonpolymorphic MHC class II determinants [22]. DA6.231 was provided by Dr. K. Guy (Edinburgh, UK) and is a broad class II MoAb that reacts with DP and DR but not with DQ [23]. IVA12 was provided by Dr. D. Capra (Dallas, TX) and reacts with DP [24]. OKT3 (anti-CD3) was obtained from Ortho (Oss, The Netherlands). MoAbs specific for CD2 (CLB-T11.2/1) and CD25 (CLB-IL-2R/ 1), provided by Dr. R.A.W. van Lier, were produced at the CLB, Amsterdam.
Indirect Immunofluorescence Assay Virus-infected and noninfected T lymphoblasts and B-cell lines were incubated for 30 min with saturating amounts of MoAb directed against monomorphic determinants on class I and class II molecules. Following two wash steps with phosphate-buffered saline (PBS) containing 0.5% (v/v) bovine serum albumin (BSA) and supplemented with 0.02% (w/v) sodium azide (PBS-BSA), the cells were incubated with fluorescein-isothiocyanate (FITC)-conjugated goat-antimouse antibodies for 30 min. Cells were washed three times with PBS-BSA and analyzed by flow cytometry (Coulter Epics-C cytofluorometer, Coulter Electronics, Hialeah, FL, or FACSCAN, Becton-Dickinson, Mountain View, CA).
Biochemical Analysis of MHC Antigens EBV-transformed B cells, PHA-stimulated T lymphoblasts, and T cells from a single individual were infected with influenza virus. 5 × 106 cells of each cell type were labeled with 50 t~Ci 35S-methionine in methionine-free RPMI prior to, or immediately after, infection. Likewise, influenza virus-infected cells (18 hr postinfection) as well as noninfected cells (5 × 106 cells for each condition) were surface-iodinated using lactoperoxidase as described [25]. After cell lysis using
202
F.T.M. Rotteveel et al NP-40 lysis mix, 1 mM 2-deoxysialic acid was added to inhibit possible neuraminidase activity [26]. Immunoprecipitations were performed out of equivalent amounts of TCA-precipitable radioactivity [27] using the MoAb W6/32 for class I antigens [21] and a rabbit polyclonal class II serum for class II [28]. Half o f the immunoprecipitates were treated with C. perfringens neuraminidase (Sigma, type VIII) as described [29] and analyzed by one-dimensional isoelectric focusing (ID-IEF) [29].
Pulse-Chase Analysis of MHC Antigens The pulse-chase analysis was performed essentially as described [27]. For this analysis, the EBV-transformed B-cell line Krij was used, since this line has been well studied with respect to class I biogenesis and transport [27]. Briefly, 15 x 106 EBV-transformed B cells were infected with influenza virus, and 15 × t() '~ noninfected cells were used as control. Then, 18 hr postinfection, cells were maintained for 0.5 hr in methionine-free RPMI-1640, followed by a 15-rain pulse with 100/.~Ci ~S-methionine. Incorporation of label was stopped through addition o f nonradioactive methionine to a final concentration of 1 mM. At time points 0, 0.5, 1, 3, and 8 hr, a sample of 3 x 106 cells was taken, lysed, and 2-deoxysialic acid was added to a final concentration of I mM. Immunoprecipitation using the MoAb W6/32, neuraminidase treatment, and 1D-IEF analysis were performed as described [29].
RESULTS Class II M H C determinants are only expressed on a small percentage of resting T lymphocytes [30]. After activation with PHA, a percentage of the CD3 ~ cells is positive for M H C class II determinants, but low compared to the expression on B-cell lines (Figure 1, Table 1). The effect of influenza virus infection on the binding of anti-MHC MoAb was investigated on T lymphocytes, PHA-stimulated T lymphoblasts, and E B V transformed B cells. Lymphocytes were infected for 1 hr with influenza virus, washed, and cultured at 37°C for 18 hr. Infection with influenza virus caused a two- to threefold increase in the binding of anti-class II MoAb on activated T cells (Table 1). N o t only was the percentage of positive cells increased but also the amount of M H C class II-specific antibodies bound per cell was enhanced (Table 1). This effect was observed with several anti-class II-specific MoAbs on T cells from six different donors. As shown (Figure 1), a similar increase in anti-class II MoAb binding was seen with different strains of influenza virus. This increase was already detectable 4 h postinfection (Figure 1). When unstimulated T cells were infected with influenza virus, an increase of anti-class II MoAb binding was also observed, whereas, interestingly, infection with influenza virus of B-cell lines had no effect on anti-MHC class II MoAb binding (Figure t). The binding of anti-MHC class I MoAb on both activated T cells and B-cell lines was not altered by influenza virus infection, whereas the binding of anti-MHC class ! MoAb to resting infected T cells was enhanced (Figure 2). W6/32 binds already strongly to activated T cells and B-cell lines. Also the binding o f anti-MHC class II MoAb to B-cell lines is very strong. This could explain why influenza virus infection did not result in further enhancement of the binding of these anti-MHC MoAbs on these cells. However, on these cells also the M H C molecules were altered biochemically--as shown by analysis of IEF--after influenza virus
Influenza Changes Cell-Surface Glycoproteins
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FIGURE 1 Binding of anti-MHC class II antibodies on T and B cells analyzed by immunofluorescence employing monoclonal antibodies R3E2, IVA12, and DA6.231 with a Coulter Epics-C. Panels (A)-(E) show the fluorescence intensity, expressed on a three-decade log scale for T lymphoblasts: noninfected (A), influenza X79 infected for 18 h (B), influenza A / H K infected for 18 h (C), influenza B infected for 18 h (D), and influenza X79 infected for 4 h (E). Panels (F) and (G) show the fluorescence intensity for B-cell lines: noninfected (F) and influenza X79 infected for 18 h (G). The dotted lines indicate the background fluorescence obtained using the second reagent alone.
incubation (see below). The increase in binding of M H C antibodies was specific, since upon influenza virus infection no such increase was observed for several other MoAbs directed against n o n - M H C surface antigens (Figure 2). Could the enhanced expression of class II antigens be due to interferons produced by virus-infected cells [31]? Recombinant human IFN-c~ (100, 1000, 10,000 U/ml) and/or rhIFN-~/ (10, 100 U/ml) added alone or in combination during 18 hr to cultures of noninfected, activated lymphocytes did not affect the binding of a n t i - M H C M o A b (Table 2). Moreover, antibodies directed against
204
F, T. M. Rotteveel et al.
TABLE 1
T h e effect of influenza virus on M H C expression on PHA-activated T cells OKT3"
D o n o r G23 Noninfected Influenza X 7 9 (18 h) Influenza A / H K (18 h) lnfluenza B (18 h) Influenza X 7 9 (4 h) D o n o r K43 Noninfected Influenza X 7 9 (18 h) Influenza A / H K (18 h) Influenza B (18 h) Influenza X 7 9 (4 h)
R3E2"
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IVA12'
%b
MFF
%
MFI
%
MFI
%
MFI
9~
MFI
94 96 96 97 96
20 21 2(} 18 15
99 99 98 99 100
312 321 312 265 244
77 92 90 94 90
21 ~6 32 60 2v
79 93 92 94 90
13 24 18 20 14
8q 9~ 94 92 95
2i .i2 ~L ~6 i79
95 95 95 96 96
26 22 23 22 17
99 99 99 98 99
296 304 338 321 238
68 91 91 95 89
12 29 20 40 16
67 92 91 96 88
9 19 13 19 10
69 82 9~ 92 96
/ ', ~(I 21 b:{ i(,
W6/32 is a MoAb directed against class I determinants. R3E2, DA6.231, and IVAI2 are MoAbs directed against class I! determinants. OKT3 is a MoAb directed against CD3. b Percentage of positive cells. c Membrane fluorescence intensity was analyzed by flow cytometry and is expressed as mean fluorescence intensity (MFI).
TABLE 2
Effect o f I F N - a and/or IFN-y on class I and II antigen expression on activated T lymphocytes and monocytes Class I W6/32 MoAb
Activated T lymphocytes + 10 U IFN-9, + 100 U IFN- 7 + 100 U I F N - a 2 b + 1000 U I F N - a 2 b + 10000 U IFN-c~2b Influenza X 7 9 infection + anti-IFN-7 + anti-IFN-a2b + anti-IFN-ce2b,5, Monocytes + 10 U IFN-~, + 100 U IFN-'y + 10 U IFN-~/ + anti-IFN-y + 1000 U IFN-c,2b + 10000 U I F N - a 2 b + 1000 U I F N - a 2 b + anti-IFN-oe2b ° Percentage of positive cells in indirect fluorescence. b Mean fluorescence intensity.
Class 11 R3E2
%~
MFI ~
~"
MFP
99 99 99 99 99 99 98 97 98 98 100 100 100 100 100 100 100
244 177 181 181 163 186 272 219 225 244 54 97 100 48 95 118 54
73 77 77 71 Vl 76 90 94 94 96 95 98 97 99 97 98 98
I~,i ]] i 10 L1 i ~4 i,~, :i 7 4! 10 t00 118 ,2 2(~ ~'>~ 20
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log fluorescence intensity FIGURE 2 Phenotypic analysis of noninfected resting T cells (unbroken lines) and influenza virus-infected T cells (dotted lines) with a FACSCAN. The cells were indirectly stained against CD3 (OKT3), CD2 (6G4), and CD25 (TB30). MHC class I (W6/32) and MHC class II (R3E2) with medium ( - ) as control. The fluorescence intensity is expressed on a log scale.
I F N - ~ and -~ did not inhibit the increase of anti-MHC class II M o A b binding on virus-infected cells (Table 2). In control experiments using monocytes, I F N - a and -~ were capable o f causing an increase in M H C class II expression during 18 hr of incubation. This increase was blocked by anti-IFN-~ and anti-IFN-~ antibodies, respectively (Table 2). Expression o f M H C antigens has been reported to depend on the activation state o f the lymphoid cells [32]. If true, the increased M H C expression observed in our experiments could reflect a change in the activation state of the infected cells caused by influenza virus [33]. However, infection of resting T cells with influenza virus enhanced binding of MHC-specific antibodies, whereas the binding o f other antibodies recognizing characteristic activation markers as CD2 and CD25 (IL-2R) was not altered by influenza virus infection (Figure 2). Moreover, we showed that the different influenza virus strains did not have mitogenic activity (Table 3). Thus, we conclude that virus infection per se does
206
F.T.M. Rotteveet et aL TABLE 3
Influenza virus induces no proliferation of lymphocytes Cell p r o l i f e r a t i o n ( c p m y
Stimulus
Donor
Medium Influenza Influenza Influenza Influenza P H A '~
X79-infected/' A/HK added' X79 added' B added
K56 -~7 79 72 103 158 12,477
± + ± ± -+ ±
HS(.I 15 18 11 13 23 395
563 504 (~40 454 922 ~'5,894
:;-. i317 -~ 215 ± I 0: ~ 29 ~ IOl ± 535
"Cell proliferation measured by 3H-thymidine incorporation at day 3 (mean of triplicate -+ SD). PBMCs were incubated for 90 min with influenza X79. After infection, cells were washed and resuspended lr~ RPMI containing 10% FCS. ' 5/zl influenza virus A / H K , X79, or B was added to peripheral blood mononuclear cells for ~2 hr; ~he last 2i hr~ 3H-thymidine was added. d P H A was added at the final concentration of 5 I.Lg/ml.
not result in significant T-cell activation when analyzed by the protocol described in "Materials and Methods." Biochemical analysis o f M H C class I and II antigens from surface-iodinated and from 35S-methioninedabeled human T and B cells was performed to investigate whether virus infection had caused a change in their biochemical properties. 1D-IEF of M H C antigens immunoprecipitated with monomorphic anti-MHC M o A b showed similar isoelectric point (IEP) values for M H C antigens from unstimulated T cells, T lymphoblasts, and B-cell lines. After influenza virus infection, the IEP of both class I and class tI M H C antigens is changed in T cells as well as in B cells (Figure 3). The IEP is shifted upward to a higher pI value, consistent with removal of sialic acids. N o difference in the amount of the M H C antigens, as assayed by quantitative immunoprecipitation from equal amounts of radiolabeled lysates, was observed after influenza virus infection (see Figures 3 and 5). Removal of sialic acids was not restricted to M H C antigens, because the IEP o f the transferrin receptor was also changed (data not shown). It has been shown already [34] that influenza virus infection affects the binding o f MHC-specific antibodies due to the effect of viral neuraminidase, but the molecular basis was not elucidated. Therefore, we analyzed the effect of C. perfringens neuraminidase treatment on T and B cells. The results revealed a similar shift in IEP on both M H C class I and class II antigens as was caused by influenza virus infection (Figure 3). Immunofluorescence analysis showed that after neuraminidase treatment of resting T cells and T lymphoblasts, the binding o f anti-MHC class II MoAb increased in the same way as it did after influenza virus infection, whereas the binding of other antibodies was not changed (Figure 4). Neuraminidase treatment of B-cell lines did not alter the binding of anti-MHC class II MoAbs, a result similar to that obtained after influenza virus infection (data not shown). Therefore, the effect of viral infection on the binding o f anti-MHC MoAbs is caused by the action o f the viral neuraminidase on M H C antigens rather than an increase in number of M H C antigens per cell. Increased binding of anti-MHC antibodies is not likely to be the consequence of better accessibility to antibodies o f the cell surface as a whole due to removal of sialic acids, because the binding of other antibodies is not affected (Figure 4). The effect o f virus infection on the IEP of M H C antigens was most pronounced in B-cell lines and resting T cells. In virus-infected, activated T lymphoblasts, a shift
Influenza Changes Cell-Surface Glycoproteins
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F I G U R E 3 Biochemical analysis by IEF of MHC molecules from B-cell lines, resting T cells, and PHA-stimulated T lymphoblasts, noninfected ( - ) and influenza virus-infected (+). Lysates were prepared from iodinated or 35S-methionine-labeled cells. Immunoprecipitation using the anti-MHC class I MoAb W6/32 (A) or an anti-class II serum [25] (B) was performed out of an equal amount of TCA-precipitate radioactivity. The effect of C. perfringens neuraminidase ( N A N A ) on the IEF pattern of MHC antigens was shown in the right part of (A) and (B). Anode is at the bottom.
208
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log fluorescence intensity FIGURE 4 Expression of cell surface antigens on influenza virus-infected (FLU) and neuraminidase (NANA)-treated T lymphocytes. The dotted lines indicate the flu()rescence without infection of neuraminidase treatment. The cells were incubated with 2 U/ml C. perfringens neuraminidase (Sigma, type VIII) in PBS for 30 rain at 37°C. Cells were stained for surface immunofluorescence with MoAb to CD3 (OKT3), MHC class II (R3E2), and using the second reagent alone ( - ) and analyzed by flow microfluorometry with a FACSCAN. The fluorescence intensity is expressed on a log scale.
in IEP was nevertheless clearly detectable [Figure 3(B)] and, furthermore, these infected T lymphoblasts were excellent targets for virus-specific cytotoxic T lymphocytes (data not shown). Pulse-chase experiments were carried out to investigate the biosynthesis of M H C antigens in virus-infected and control cells. In control cells, both M H C class I and class II antigens are modified such that the high-mannose glycans, transferred cotranslationally, are modified to complex type N-linked glycans in the Golgi apparatus. These modifications include the addition of sialic acids, a process that is readily visualized by a decrease in pI (Figure 5). In influenza virus-infected cells, the addition of sialic acids can still be observed (compare 0 and 0.5 h, Figure 5), but even after 30 min of chase, M H C class I molecules carry fewer sialic acids than noninfected controls. It is significant that appearance at the cell surface of, for example, H L A - A 2 does not become detectable until between 40 and 60 min of chase [27]; thus, this reduction in sialic acid content must take place en route to the cell surface. Ultimately, complete removal o f sialic acids is the result (compare 8-hr time point and 8-hr time point after digestion with neuraminidase, Figure 5). This result is consistent with exposure of class I molecules already at an early stage of intracellular transport (30 min) [35] as well as after their arrival at the cell surface to the viral neuraminidase.
Influenza Changes Cell-Surface Glycoproteins
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® FIGURE 5 Pulse-chase analysis of class I antigens of the control and influenza virus (+flu)-infected B-cell line Krij. Cells were pulse-labeled for 15 min. The different chase times (hr) are indicated. Neuraminidase (N) treatment was carried out after 8 h (lane indicated 8+N). The position of the different class I heavy chains is indicated on the right, from cathodic to anodic HLA-A2, 32m, B35, A l l , and B27K, respectively, while the different sialiated species are indicated on the left again for A2, B35, A11, and B27K from cathode ( - ) to anode (+). The indexes refer to the 1 and 2 sialic acid-containing species, respectively. An included normal rabbit serum (NRS) control demonstrated specificity of the experiment.
DISCUSSION A number of viruses have been shown to modulate the levels of M H C class I antigens in the cells they infect or transform. In several instances, a reduction was observed, such as in Ad5-transformed and Ad2-infected cells [8]. In the former, this process is controlled at the level of the M H C transcript, whereas in the latter association with a viral polypeptide leads to arrest of M H C class I molecules in the endoplasmic reticulum [ 14]. Other examples of viruses that cause a reduction in the amount o f M H C antigens include poliomyelitis virus and vesicular stomatitis virus [10]. Cells infected with mouse hepatitis virus [13] and simian immunodeficiency virus [12] show an increase in expression of M H C class II antigens, whereas human immunodeficiency virus infection results in reduced amounts o f class II determinants [11]. Recently, increased binding of H L A - D R specific antibodies was shown for measles virus-infected T lymphoblasts [36]. We detected increased levels of M H C antigens on cells infected with influenza virus. This is not due to a net increase in the amount of M H C antigens, but to increased binding of anti-MHC MoAbs to M H C molecules. This increase in binding is satisfactorily explained by removal of sialic acids from the M H C
210
F.T.M. Rotteveel et al. antigens by the viral neuraminidase. Interestingly, this effect appears to be rather specific for anti-MHC antibodies, because the binding of a number of other MoAbs recognizing surface molecules was not affected by influenza virus infection. The presence of sialic acids, which are characterized by their net negative charge at neutral pH, could interfere with interactions between surface receptors and their counterstructures in a more general way. In our study~ anti-MHC antibodies serve as illustrative examples, but the findings reported here could be extended to other interactions as well. Because the parameters listed (e.g., viral infection, glycosylation) influence the usual measurements of antigen density, one should be careful to quantitate expression of surface molecules by FACS analysis only. Pulse-chase experiments are consistent with the notion that removal of sialic acids takes place both intracellutarly and at the cell surface. N o t unexpectedly~ neuraminidase action was not restricted to M H C antigens, because a nonrelated surface structure, the transferrin receptor, was also desialated almost completely (unpublished observation). M H C molecules are essential for the presentation of antigens to T cells, it is possible that altered M H C determinants, by removal of sialic acids in influenza virus-infected T cells, can affect antigen presentation. Recently, it was demonstrated [ 3 7 - 4 0 ] that desialated cells were better APCs than untreated cells. Whereas the virus itself requires neuraminidase for infectivity, by coincidence its action may be beneficial to the host in that it can increase the efficiency of APCs. It is already reported that T tymphobtasts are able to function as APCs [41,42]~ Finally, the removal of sialic acids of lymphocytes by viral neuraminidase in vivo may well affect the traffic of lymphocytes, with pathological consequences [43]~ ACKNOWLEDGMENTS
This study was supported in part by the Foundation for Medical Research MEDIGON, which is subsidized by the Netherlands Organization for Scientific Research (NWO) (grant 13-04-05) and in part by the Queen Wilhelmina Fund (grant NKI-85-9). The authors wish to thank Mrs. Jetty Gerritsen for typing the manuscript. REFERENCES 1. Janeway CA, Bottomly K, Babich J, Conrad P, Conzen S, Jones B, Kaye J, Katz M~ McVay L, Murphy DB, Tite J: Quantitative variation in Ia antigen expression plays central role in immune regulation. Immunol Today 5:99, 1984. 2. Matis LA, Glimcher LH, Paul WE, Schwartz RH: Magnitude of response oi histocompatibility-restricted T-cell clones is a function of the product of the concentrations of antigen and Ia molecules. Proc Natl Acad Sci USA 80:6019, 1983. 3. Schwartz RH: T-lymphocyte recognition of antigen in association with gene products of the major histocompatibility complex. Ann Rev Immunol 3:237, 1985. 4. Henry C, Chan EL, Kodlin D: Expression and function of I region products or~ immunocompetent cells. II. I region products in T-B interaction. J Immunol 119"74~L 1977. 5. Singer A, Morrissey PJ, Hathcock KS, Ahmed A, Scher I, Hodes RJ: Role of the major histocompatibility complex in T-cell activation of B-cell subpopulations. J Exp Med 154:501, 1981. 6. Flyer DC, BurakoffSJ, Failer DV: Retrovirus-induced changes in major histocompat-
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ibility complex antigen expression influence susceptibility to lysis by cytotoxic T lymphocytes. J Immunol 135:2287, 1985. 7. Matis LA, Jones PP, Murphy DP, Hedrick SM, Lerner EA, Janeway Jr CA, McNicholas JM, Schwartz RH: Immune response gene function correlates with the expression of an Ia antigen. J Exp Med 155:508, 1982. 8. Vaessen RTMJ, Houweling A, van der Eb AJ: Post-transcriptional control of class I MHC mRNA expression in adenovirus 12-transformed cells. Science 235:1486, 1987. 9. Jennings SR, Rice PL, Kloszewski ED, Anderson RW, Thompson DL, Tevethia SS: Effect of herpes simplex virus types 1 and 2 on surface expression of class I major histocompatibility complex antigens on infected cells. J Virol 56:757, 1985. 10. Haspel MV, Pellegrino MA, Lampert PW, Oldstone MBA: Human histocompatibility determinants and virus antigens: Effect of measles virus infection on HLA expression. J Exp Med 146:146, 1977. 11. Petit AJC, Terpstra FG, Miedema F: Human immunodeficiency virus infection down-regulates HLA class II expression and induces differentiation in promonocytic U937 cells. J Clin Invest 79:1883, 1987. 12. Kannagi M, Kiyotaki M, King NW, Lord CI, Letvin NL: 1987. Simian immunodeficiency virus induces expression of class II major histocompatibility complex structures on infected target cells in vitro. J Virol 61:1421, 1987. 13. Massa PT, D6rries R, ter Meulen V: Viral particles induce Ia antigen expression on astrocytes. Nature 320:543, 1986. 14. P~i~ibo S, Nilsson T, Peterson PA: Adenoviruses of subgenera B, C, D, and E modulate cell-surface expression of major histocompatibility complex class I antigens. Proc Natl Acad Sci USA 83:9665, 1986. 15. Fierz W, Endler B, Reske K, Wekerle H, Fontana A: Astrocytes as antigen-presenting cells. I. Induction of Ia antigen expression on astrocytes by T cells via immune interferon and its effect on antigen presentation. J Immunol 134:3785, 1985. 16. Trinchieri G, Perussia B: Immune interferon: A pleiotropic lymphokine with multiple effects. Immunol Today 6:131, 1985. 17. Wong GHW, Clark-Lewis I, Mckimm-Breschkin JL, Harris AW, Schrader JW: Interferon-y induces enhanced expression of Ia and H-2 antigens on B-lymphoid, macrophage, and myeloid cell lines. J Immunol 131:788, 1983. 18. Steeg PS, Moore RN, Johnson HM, Oppenheim JJ: Regulation of murine macrophage Ia antigen expression by a lymphokine with immune interferon activity. J Exp Med 156:1780, 1982. 19. Collins T, Korman AJ, Wake CT, Boss JM, Kappes DJ, Fiers W, Ault KA, Gimbrone Jr MA, Strominger JL, Pober JS: Immune interferon activates multiple class II major histocompatibility complex genes and the associated invariant chain gene in human endothelial cells and dermal fibroblasts. Proc Natl Acad Sci USA 81:4917, 1984. 20. Pujol-Borrell R, Todd I, Doshi M, Bottazzo GF, Sutton R, Gray D, Adolf GR, Feldmann M: HLA class-II induction in human islet cells by interferon-y plus tumor necrosis factor or lymphotoxin. Nature 326:304, 1987. 21. Parham P, Barnstable CJ, Bodmer WF: Use of a monoclonal antibody (W6/32) in structural studies of HLA-A,B,C antigens. J Immunol 123:342, 1979. 22. De Rie MA, Kabel P, Sauerwein RW, van Lier RAW, von dem Borne AEGJr, Melief CJM, Miedema F: Anti-HLA class-II monoclonal antibodies inhibit polyclonal B-cell differentiation in vitro at the accessory cell level. Eur J Immunol 17:881, 1987.
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F.T.M. Rotteveel et al. 23. Guy K, van Heyningen V, Cohen BB, Deane DL, Steel CM: Differential expression and serologically distinct subpopulations of human Ia antigens detected with monoclonal antibodies to Ia alpha and beta chains. Eur J Immunol 12:942, 1982. 24. Shaw S, Sanchez-Perez M, DeMars R: Analysis of DP region products by T cells and monoclonal antibodies: Blocking of DP-specific proliferation and cell-mediated cytotoxicity. In Albert ED (ed): Histocompatibility Testing. Berlin/Heidelberg, Springer-Verlag, 1984, p 465. 25. Phillips DR, Morrison M: The arrangement of proteins in the human erythrocyte membrane. Biochem Biophys Res Commun 40:284, 1970. 26. Meindl P, Bodo G, Palese P, Schulman J, Tuppy H. Inhibition of neuraminidase activity by derivatives of 2-deoxy-2,3-dehydro-N-acetylneuraminic acid. Virology 58:457, 1974. 27. Neefjes JJ, Ploegh HL: Allele and locus-specific differences in cell surface expression and the association of HLA class I heavy chain with ~2-microglobutin: Differential effects of inhibition of gIycosylation on class I subunit association. Eur J lmmunol 18:801, 1988. 28. Neefjes JJ, Hensen EJ, de Kroon TIP, Ploegh HL: A biochemical characterization ot feline MHC products: Unusually high expression of class II antigens on peripheral blood lymphocytes, lmmunogenetics 23:341, 1986. 29. Neefjes JJ, Breur-Vriesendorp BS, van Seventer GA, Ivanyi P, Ploegh HL: An improved biochemical method for the analysis of HLA class I antigens. Definition of new HLA class ! subtypes. Human Immunol 16:169, 1986. 30. Zier KS: Expression ofclass-II antigens by subsets of activated T cells. Cell Immunol 100:525, 1986. 31. Yamada YK, Meager A, Yamada A, Ennis FA: Human interferon alpha and gamma production by lymphocytes during the generation of influenza virus-specific cytotoxic T lymphocytes. J Gen Virol 67:2325, 1986. 32. Matsui Y, Staunton DE, Shapiro HM, Yunis EJ: Comparison of MHC antigen expression on PHA- and MLC-induced T-cell lines with that on T- and B-tymphoblastold cell lines by cell cycle dependency. Human Immunol 15:285, 1986. 33. Butchko GM, Armstrong RB, Martin WJ, Ennis FA: Influenza A viruses of the H2N2 subtype are lymphocyte mitogens. Nature 271:66, 1978. 34. Liberti PA, Hackett CJ, Askonas BA: Influenza virus infection of mouse lymphoblasts alters the binding affinity of anti-H-2 antibody: Requirement for viral neuraminidase. EurJ Immunol 9:751, 1979. 35. Fuller SD, Bravo R, Simons K: An enzymatic assay reveals that proteins destined for the apical or basolateral domains of an epithelial cell line share the same late Golgi compartments. EMBO J 4:297, 1985. 36. McChesney MB, Altman A, Oldstone MBA: Suppression of T-lymphocyte function by measles virus is due to cell cycle arrest in G1. J Immunol 140:1269, 1988. 37. Powell LD, Whiteheart SW, Hart GW: Cell surface sialic acid influences tumor cell recognition in the mixed lymphocyte reaction. J Immunol 139:262, 1987. 38. Frohman M, Cowing C: Presentation of antigen by B cells: Functional dependence on radiation dose, interleukins, cellular activation, and differential glycosylation. ] Immunol 134:2269, 1985. 39. Hirayama Y, Inaba K, Inaba M, Kato T, Kitaura M, Hosokawa T, Ikehara S, Muramatsu S: Neuraminidase-treated macrophages stimulate allogeneic CD8 ~ T cells in the presence of exogenous interleukin 2. J Exp Med 168:1443, 1988.
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40. Boog CJP, Neefjes JJ, Boes J, Ploegh HL, Melief CJM: Immune responses restored by alteration in carbohydrate chains of histocompatibility antigens on antigen presenting cells. Eur J Immunol, 19:537, 1989. 41. Kabelitz D, Enssle K-H, Fleischer B, Reimann J: Antigen-presenting T cells. II. Clonal responses of alloreactive and virus-specific self-restricted human cytotoxic T-cell responses stimulated by T lymphoblasts. J Immunol 138:45, 1987. 42. Brown MF, Cook RG, Van M, Rich RR: Cloned human T cells synthesize Ia molecules and can function as antigen presenting cells. Human Immunol 11:219, 1984. 43. Woodruff JJ, Gesner BM: The effect of neuraminidase on the fate of transfused lymphocytes. J Exp Med 129:551, 1969.
A B S T R A C T : The effect of influenza virus infection on the expression of major histocompatibility complex (MHC) antigens was investigated. Infection with influenza virus resulted in an increase of the binding of anti-MHC class I and class II antibodies to resting T cells. The binding of anti-MHC class II antibodies to activated T cells was increased approximately threefold. The binding of anti-MHC class I and class II antibodies to Epstein-Barr virus -transformed B cells appeared unaffected after influenza virus infection. Recombinant human interferon-~ and/or -7 added to T cells did not enhance the binding of anti-MHC antibodies. Biochemical analysis revealed no increase in the amount of class I and class II antigens as a consequence of viral infection, but a marked decrease in sialic acid content was found, most probably caused by the viral neuraminidase. Pulse-chase experiments suggest that the viral neuraminidase can catalyze the removal of sialic acids both en route to and at the cell surface. The absence of sialic acid residues can explain the increased binding of anti-MHC antibodies, because neuraminidase (clostridium perfringens) treatment of T and Epstein-Barr virus-transformed B cells resulted in a shift in both isoelectric point and antibody binding similar to that observed after influenza virus infection. ABBREVIATIONS
APC EBV FCS IFN MHC
antigen-presenting cells Epstein-Barr virus fetal calf serum interferon major histocompatibility complex
MoAb 1D-IEF PHA
monoclonal antibody one-dimensional isoelectric focusing phytohemagglutinin
INTRODUCTION
The products encoded by the major histocompatibility complex (MHC) guide lymphocytes in their interactions with antigen-presenting cells (APCs) or target cells. CD4 ÷ cells predominantly utilize M H C class II molecules as restriction From the Central Laboratory of the Netherlands Red Cross Blood Transfusion Serviceand the Laboralory for Experimental and Clinical Immunology, University of Amsterdam (F.T.M.R., CJ.L.) and the Netherlands Cancer Institute, Amsterdam, the Netherlands (J.J.N., H.L.P.). Address reprint requests to Dr. CorneliusJ. Lucas, c/o Publication Secretariat, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, P.O. Box 9406, 1006 AK Amsterdam, the Netherlands. ReceivedJanuary 17~ 1989; revisedMay 8. 1989.
Human Immunology26, 199-213 (1989) © AmericanSocietyfor Histocompatibilityand Immunogenetics,1989
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F.T.M. Rotteveel et al. elements, whereas CD8 ÷ T cells preferentially recognize antigens presented by M H C class I determinants. It has been shown that the level at which M H C products are expressed on APCs is an important parameter in determining the ability to present antigen to T lymphocytes [1-3] and in T-/B-cell interactions [4,5]. The absence of M H C class I antigens renders a cell resistant to attack by cytotoxic CD8 ÷ T cells. Similarly, a cell devoid of class II antigens cannot present a processed foreign antigen to a CD4 + T cell. A dose-response relationship has been established for the amount of M H C class I antigen, and the ability to induce a cytotoxic T lymphocyte response in vitro [6]. A threshold level of M H C class l I antigens is also required to trigger an immune response, the magnitude of which increases concomitantly with class II antigen expression [7]. Thus, the levels of M H C antigen determine whether or not stimulation of responder cells wilt ensue. Several viruses have been shown to modulate the level of MHC determinants in APCs or target cells. Certain viruses abrogate expression of M H C class [ [8-10] and/or class II antigens [11], whereas others cause an increase in levels of M H C class I [6] or class II [12,13] determinants. This control of expression of M H C antigens is exerted at different levels. A decrease in expression may bc caused by transcriptional and/or posttranscriptional processes [8,14], Examples of both types have been documented. An increase in expression may be due to the production of and response to interferons, potent cytokines that generally increase levels of class I [interferon (IFN)-~, -/3, -5~] and class II (IFN-50 on many susceptible cell types [15-19], an effect that may be potentiated by tumor necrosis factor [20]. In the present report, we describe the effect of influenza virus infection on M H C class I and class II molecules. We show that removal of sialic acids by the virally encoded neuraminidase results in increased binding of anti-MHC monoclonal antibodies (MoAbs), the actual levels of MHC-encoded potypeptides being identical in virus-infected and control cells. Analysis of the biosynthesis of M H C products in virus-infected cells suggests that removal of sialic acids takes place both intracellularly and at the cell surface.
MATERIALS AND METHODS Cells Peripheral blood mononuclear cells (PBMCs) were obtained by Ficoll-Hypaque density centrifugation of peripheral blood from healthy donors. T cells were isolated by sheep erythrocyte (E)-rosette sedimentation. Activated cells were prepared by culture of PBMCs in the presence of phytohemagglutinin (PHA) (5 /.*g/ml; Gibco Laboratories, Grand Island, NY) for 3 days. B-lymphoblastoid cell lines were established by infection of B cells with Epstein-Barr virus (EBV). The B-cell lines were grown in RPMI-1640 containing I0% fetal calf serum (FCS). The monocyte population was obtained by countercurrent centrifugal elutriation. Interferon Recombinant human IFN-o~2b and IFN-5/were generously provided by Dr. van der Meide (TNO, Rijswijk, The Netherlands). The anti-IFN antibodies were used at a concentration of about 3000 neutralizing U/ml.
Influenza Changes Cell-Surface Glycoproteins
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Virus Influenza A / H K [A/Hong Kong/8/68-X-31 (H3N2)] was a generous gift from Prof. dr. N. Masurel (Erasmus University, Rotterdam, The Netherlands). Influenza A/X79 and influenza B/Singapore were gifts from Duphar (Weesp, The Netherlands).
Virus Infection of the Cells Activated T cells (cultured for 2 days with PHA), unstimulated T cells, or B cells were incubated for 90 min with influenza virus. After infection, cells were washed and resuspended in RPMI containing 10% FCS and incubated at 37°C for 18 hr.
Monoclonal Antibodies The MoAb directed against HLA class I determinants, W6/32 (Hb 95), was obtained through the American Type Culture Collection (ATCC, Rockville, MD) [21]. MHC class II-specific MoAbs, R3E2, DA6.231, and IVA12 were used. R3E2 was provided by Dr. M. A. de Rie [Central Laboratory of the Red Cross Blood Transfusion Service (CLB), Amsterdam] and recognizes nonpolymorphic MHC class II determinants [22]. DA6.231 was provided by Dr. K. Guy (Edinburgh, UK) and is a broad class II MoAb that reacts with DP and DR but not with DQ [23]. IVA12 was provided by Dr. D. Capra (Dallas, TX) and reacts with DP [24]. OKT3 (anti-CD3) was obtained from Ortho (Oss, The Netherlands). MoAbs specific for CD2 (CLB-T11.2/1) and CD25 (CLB-IL-2R/ 1), provided by Dr. R.A.W. van Lier, were produced at the CLB, Amsterdam.
Indirect Immunofluorescence Assay Virus-infected and noninfected T lymphoblasts and B-cell lines were incubated for 30 min with saturating amounts of MoAb directed against monomorphic determinants on class I and class II molecules. Following two wash steps with phosphate-buffered saline (PBS) containing 0.5% (v/v) bovine serum albumin (BSA) and supplemented with 0.02% (w/v) sodium azide (PBS-BSA), the cells were incubated with fluorescein-isothiocyanate (FITC)-conjugated goat-antimouse antibodies for 30 min. Cells were washed three times with PBS-BSA and analyzed by flow cytometry (Coulter Epics-C cytofluorometer, Coulter Electronics, Hialeah, FL, or FACSCAN, Becton-Dickinson, Mountain View, CA).
Biochemical Analysis of MHC Antigens EBV-transformed B cells, PHA-stimulated T lymphoblasts, and T cells from a single individual were infected with influenza virus. 5 × 106 cells of each cell type were labeled with 50 t~Ci 35S-methionine in methionine-free RPMI prior to, or immediately after, infection. Likewise, influenza virus-infected cells (18 hr postinfection) as well as noninfected cells (5 × 106 cells for each condition) were surface-iodinated using lactoperoxidase as described [25]. After cell lysis using
202
F.T.M. Rotteveel et al NP-40 lysis mix, 1 mM 2-deoxysialic acid was added to inhibit possible neuraminidase activity [26]. Immunoprecipitations were performed out of equivalent amounts of TCA-precipitable radioactivity [27] using the MoAb W6/32 for class I antigens [21] and a rabbit polyclonal class II serum for class II [28]. Half o f the immunoprecipitates were treated with C. perfringens neuraminidase (Sigma, type VIII) as described [29] and analyzed by one-dimensional isoelectric focusing (ID-IEF) [29].
Pulse-Chase Analysis of MHC Antigens The pulse-chase analysis was performed essentially as described [27]. For this analysis, the EBV-transformed B-cell line Krij was used, since this line has been well studied with respect to class I biogenesis and transport [27]. Briefly, 15 x 106 EBV-transformed B cells were infected with influenza virus, and 15 × t() '~ noninfected cells were used as control. Then, 18 hr postinfection, cells were maintained for 0.5 hr in methionine-free RPMI-1640, followed by a 15-rain pulse with 100/.~Ci ~S-methionine. Incorporation of label was stopped through addition o f nonradioactive methionine to a final concentration of 1 mM. At time points 0, 0.5, 1, 3, and 8 hr, a sample of 3 x 106 cells was taken, lysed, and 2-deoxysialic acid was added to a final concentration of I mM. Immunoprecipitation using the MoAb W6/32, neuraminidase treatment, and 1D-IEF analysis were performed as described [29].
RESULTS Class II M H C determinants are only expressed on a small percentage of resting T lymphocytes [30]. After activation with PHA, a percentage of the CD3 ~ cells is positive for M H C class II determinants, but low compared to the expression on B-cell lines (Figure 1, Table 1). The effect of influenza virus infection on the binding of anti-MHC MoAb was investigated on T lymphocytes, PHA-stimulated T lymphoblasts, and E B V transformed B cells. Lymphocytes were infected for 1 hr with influenza virus, washed, and cultured at 37°C for 18 hr. Infection with influenza virus caused a two- to threefold increase in the binding of anti-class II MoAb on activated T cells (Table 1). N o t only was the percentage of positive cells increased but also the amount of M H C class II-specific antibodies bound per cell was enhanced (Table 1). This effect was observed with several anti-class II-specific MoAbs on T cells from six different donors. As shown (Figure 1), a similar increase in anti-class II MoAb binding was seen with different strains of influenza virus. This increase was already detectable 4 h postinfection (Figure 1). When unstimulated T cells were infected with influenza virus, an increase of anti-class II MoAb binding was also observed, whereas, interestingly, infection with influenza virus of B-cell lines had no effect on anti-MHC class II MoAb binding (Figure t). The binding of anti-MHC class I MoAb on both activated T cells and B-cell lines was not altered by influenza virus infection, whereas the binding of anti-MHC class ! MoAb to resting infected T cells was enhanced (Figure 2). W6/32 binds already strongly to activated T cells and B-cell lines. Also the binding o f anti-MHC class II MoAb to B-cell lines is very strong. This could explain why influenza virus infection did not result in further enhancement of the binding of these anti-MHC MoAbs on these cells. However, on these cells also the M H C molecules were altered biochemically--as shown by analysis of IEF--after influenza virus
Influenza Changes Cell-Surface Glycoproteins
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incubation (see below). The increase in binding of M H C antibodies was specific, since upon influenza virus infection no such increase was observed for several other MoAbs directed against n o n - M H C surface antigens (Figure 2). Could the enhanced expression of class II antigens be due to interferons produced by virus-infected cells [31]? Recombinant human IFN-c~ (100, 1000, 10,000 U/ml) and/or rhIFN-~/ (10, 100 U/ml) added alone or in combination during 18 hr to cultures of noninfected, activated lymphocytes did not affect the binding of a n t i - M H C M o A b (Table 2). Moreover, antibodies directed against
204
F, T. M. Rotteveel et al.
TABLE 1
T h e effect of influenza virus on M H C expression on PHA-activated T cells OKT3"
D o n o r G23 Noninfected Influenza X 7 9 (18 h) Influenza A / H K (18 h) lnfluenza B (18 h) Influenza X 7 9 (4 h) D o n o r K43 Noninfected Influenza X 7 9 (18 h) Influenza A / H K (18 h) Influenza B (18 h) Influenza X 7 9 (4 h)
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%
MFI
%
MFI
%
MFI
9~
MFI
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20 21 2(} 18 15
99 99 98 99 100
312 321 312 265 244
77 92 90 94 90
21 ~6 32 60 2v
79 93 92 94 90
13 24 18 20 14
8q 9~ 94 92 95
2i .i2 ~L ~6 i79
95 95 95 96 96
26 22 23 22 17
99 99 99 98 99
296 304 338 321 238
68 91 91 95 89
12 29 20 40 16
67 92 91 96 88
9 19 13 19 10
69 82 9~ 92 96
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TABLE 2
Effect o f I F N - a and/or IFN-y on class I and II antigen expression on activated T lymphocytes and monocytes Class I W6/32 MoAb
Activated T lymphocytes + 10 U IFN-9, + 100 U IFN- 7 + 100 U I F N - a 2 b + 1000 U I F N - a 2 b + 10000 U IFN-c~2b Influenza X 7 9 infection + anti-IFN-7 + anti-IFN-a2b + anti-IFN-ce2b,5, Monocytes + 10 U IFN-~, + 100 U IFN-'y + 10 U IFN-~/ + anti-IFN-y + 1000 U IFN-c,2b + 10000 U I F N - a 2 b + 1000 U I F N - a 2 b + anti-IFN-oe2b ° Percentage of positive cells in indirect fluorescence. b Mean fluorescence intensity.
Class 11 R3E2
%~
MFI ~
~"
MFP
99 99 99 99 99 99 98 97 98 98 100 100 100 100 100 100 100
244 177 181 181 163 186 272 219 225 244 54 97 100 48 95 118 54
73 77 77 71 Vl 76 90 94 94 96 95 98 97 99 97 98 98
I~,i ]] i 10 L1 i ~4 i,~, :i 7 4! 10 t00 118 ,2 2(~ ~'>~ 20
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I F N - ~ and -~ did not inhibit the increase of anti-MHC class II M o A b binding on virus-infected cells (Table 2). In control experiments using monocytes, I F N - a and -~ were capable o f causing an increase in M H C class II expression during 18 hr of incubation. This increase was blocked by anti-IFN-~ and anti-IFN-~ antibodies, respectively (Table 2). Expression o f M H C antigens has been reported to depend on the activation state o f the lymphoid cells [32]. If true, the increased M H C expression observed in our experiments could reflect a change in the activation state of the infected cells caused by influenza virus [33]. However, infection of resting T cells with influenza virus enhanced binding of MHC-specific antibodies, whereas the binding o f other antibodies recognizing characteristic activation markers as CD2 and CD25 (IL-2R) was not altered by influenza virus infection (Figure 2). Moreover, we showed that the different influenza virus strains did not have mitogenic activity (Table 3). Thus, we conclude that virus infection per se does
206
F.T.M. Rotteveet et aL TABLE 3
Influenza virus induces no proliferation of lymphocytes Cell p r o l i f e r a t i o n ( c p m y
Stimulus
Donor
Medium Influenza Influenza Influenza Influenza P H A '~
X79-infected/' A/HK added' X79 added' B added
K56 -~7 79 72 103 158 12,477
± + ± ± -+ ±
HS(.I 15 18 11 13 23 395
563 504 (~40 454 922 ~'5,894
:;-. i317 -~ 215 ± I 0: ~ 29 ~ IOl ± 535
"Cell proliferation measured by 3H-thymidine incorporation at day 3 (mean of triplicate -+ SD). PBMCs were incubated for 90 min with influenza X79. After infection, cells were washed and resuspended lr~ RPMI containing 10% FCS. ' 5/zl influenza virus A / H K , X79, or B was added to peripheral blood mononuclear cells for ~2 hr; ~he last 2i hr~ 3H-thymidine was added. d P H A was added at the final concentration of 5 I.Lg/ml.
not result in significant T-cell activation when analyzed by the protocol described in "Materials and Methods." Biochemical analysis o f M H C class I and II antigens from surface-iodinated and from 35S-methioninedabeled human T and B cells was performed to investigate whether virus infection had caused a change in their biochemical properties. 1D-IEF of M H C antigens immunoprecipitated with monomorphic anti-MHC M o A b showed similar isoelectric point (IEP) values for M H C antigens from unstimulated T cells, T lymphoblasts, and B-cell lines. After influenza virus infection, the IEP of both class I and class tI M H C antigens is changed in T cells as well as in B cells (Figure 3). The IEP is shifted upward to a higher pI value, consistent with removal of sialic acids. N o difference in the amount of the M H C antigens, as assayed by quantitative immunoprecipitation from equal amounts of radiolabeled lysates, was observed after influenza virus infection (see Figures 3 and 5). Removal of sialic acids was not restricted to M H C antigens, because the IEP o f the transferrin receptor was also changed (data not shown). It has been shown already [34] that influenza virus infection affects the binding o f MHC-specific antibodies due to the effect of viral neuraminidase, but the molecular basis was not elucidated. Therefore, we analyzed the effect of C. perfringens neuraminidase treatment on T and B cells. The results revealed a similar shift in IEP on both M H C class I and class II antigens as was caused by influenza virus infection (Figure 3). Immunofluorescence analysis showed that after neuraminidase treatment of resting T cells and T lymphoblasts, the binding o f anti-MHC class II MoAb increased in the same way as it did after influenza virus infection, whereas the binding of other antibodies was not changed (Figure 4). Neuraminidase treatment of B-cell lines did not alter the binding of anti-MHC class II MoAbs, a result similar to that obtained after influenza virus infection (data not shown). Therefore, the effect of viral infection on the binding o f anti-MHC MoAbs is caused by the action o f the viral neuraminidase on M H C antigens rather than an increase in number of M H C antigens per cell. Increased binding of anti-MHC antibodies is not likely to be the consequence of better accessibility to antibodies o f the cell surface as a whole due to removal of sialic acids, because the binding of other antibodies is not affected (Figure 4). The effect o f virus infection on the IEP of M H C antigens was most pronounced in B-cell lines and resting T cells. In virus-infected, activated T lymphoblasts, a shift
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F I G U R E 3 Biochemical analysis by IEF of MHC molecules from B-cell lines, resting T cells, and PHA-stimulated T lymphoblasts, noninfected ( - ) and influenza virus-infected (+). Lysates were prepared from iodinated or 35S-methionine-labeled cells. Immunoprecipitation using the anti-MHC class I MoAb W6/32 (A) or an anti-class II serum [25] (B) was performed out of an equal amount of TCA-precipitate radioactivity. The effect of C. perfringens neuraminidase ( N A N A ) on the IEF pattern of MHC antigens was shown in the right part of (A) and (B). Anode is at the bottom.
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log fluorescence intensity FIGURE 4 Expression of cell surface antigens on influenza virus-infected (FLU) and neuraminidase (NANA)-treated T lymphocytes. The dotted lines indicate the flu()rescence without infection of neuraminidase treatment. The cells were incubated with 2 U/ml C. perfringens neuraminidase (Sigma, type VIII) in PBS for 30 rain at 37°C. Cells were stained for surface immunofluorescence with MoAb to CD3 (OKT3), MHC class II (R3E2), and using the second reagent alone ( - ) and analyzed by flow microfluorometry with a FACSCAN. The fluorescence intensity is expressed on a log scale.
in IEP was nevertheless clearly detectable [Figure 3(B)] and, furthermore, these infected T lymphoblasts were excellent targets for virus-specific cytotoxic T lymphocytes (data not shown). Pulse-chase experiments were carried out to investigate the biosynthesis of M H C antigens in virus-infected and control cells. In control cells, both M H C class I and class II antigens are modified such that the high-mannose glycans, transferred cotranslationally, are modified to complex type N-linked glycans in the Golgi apparatus. These modifications include the addition of sialic acids, a process that is readily visualized by a decrease in pI (Figure 5). In influenza virus-infected cells, the addition of sialic acids can still be observed (compare 0 and 0.5 h, Figure 5), but even after 30 min of chase, M H C class I molecules carry fewer sialic acids than noninfected controls. It is significant that appearance at the cell surface of, for example, H L A - A 2 does not become detectable until between 40 and 60 min of chase [27]; thus, this reduction in sialic acid content must take place en route to the cell surface. Ultimately, complete removal o f sialic acids is the result (compare 8-hr time point and 8-hr time point after digestion with neuraminidase, Figure 5). This result is consistent with exposure of class I molecules already at an early stage of intracellular transport (30 min) [35] as well as after their arrival at the cell surface to the viral neuraminidase.
Influenza Changes Cell-Surface Glycoproteins
209
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® FIGURE 5 Pulse-chase analysis of class I antigens of the control and influenza virus (+flu)-infected B-cell line Krij. Cells were pulse-labeled for 15 min. The different chase times (hr) are indicated. Neuraminidase (N) treatment was carried out after 8 h (lane indicated 8+N). The position of the different class I heavy chains is indicated on the right, from cathodic to anodic HLA-A2, 32m, B35, A l l , and B27K, respectively, while the different sialiated species are indicated on the left again for A2, B35, A11, and B27K from cathode ( - ) to anode (+). The indexes refer to the 1 and 2 sialic acid-containing species, respectively. An included normal rabbit serum (NRS) control demonstrated specificity of the experiment.
DISCUSSION A number of viruses have been shown to modulate the levels of M H C class I antigens in the cells they infect or transform. In several instances, a reduction was observed, such as in Ad5-transformed and Ad2-infected cells [8]. In the former, this process is controlled at the level of the M H C transcript, whereas in the latter association with a viral polypeptide leads to arrest of M H C class I molecules in the endoplasmic reticulum [ 14]. Other examples of viruses that cause a reduction in the amount o f M H C antigens include poliomyelitis virus and vesicular stomatitis virus [10]. Cells infected with mouse hepatitis virus [13] and simian immunodeficiency virus [12] show an increase in expression of M H C class II antigens, whereas human immunodeficiency virus infection results in reduced amounts o f class II determinants [11]. Recently, increased binding of H L A - D R specific antibodies was shown for measles virus-infected T lymphoblasts [36]. We detected increased levels of M H C antigens on cells infected with influenza virus. This is not due to a net increase in the amount of M H C antigens, but to increased binding of anti-MHC MoAbs to M H C molecules. This increase in binding is satisfactorily explained by removal of sialic acids from the M H C
210
F.T.M. Rotteveel et al. antigens by the viral neuraminidase. Interestingly, this effect appears to be rather specific for anti-MHC antibodies, because the binding of a number of other MoAbs recognizing surface molecules was not affected by influenza virus infection. The presence of sialic acids, which are characterized by their net negative charge at neutral pH, could interfere with interactions between surface receptors and their counterstructures in a more general way. In our study~ anti-MHC antibodies serve as illustrative examples, but the findings reported here could be extended to other interactions as well. Because the parameters listed (e.g., viral infection, glycosylation) influence the usual measurements of antigen density, one should be careful to quantitate expression of surface molecules by FACS analysis only. Pulse-chase experiments are consistent with the notion that removal of sialic acids takes place both intracellutarly and at the cell surface. N o t unexpectedly~ neuraminidase action was not restricted to M H C antigens, because a nonrelated surface structure, the transferrin receptor, was also desialated almost completely (unpublished observation). M H C molecules are essential for the presentation of antigens to T cells, it is possible that altered M H C determinants, by removal of sialic acids in influenza virus-infected T cells, can affect antigen presentation. Recently, it was demonstrated [ 3 7 - 4 0 ] that desialated cells were better APCs than untreated cells. Whereas the virus itself requires neuraminidase for infectivity, by coincidence its action may be beneficial to the host in that it can increase the efficiency of APCs. It is already reported that T tymphobtasts are able to function as APCs [41,42]~ Finally, the removal of sialic acids of lymphocytes by viral neuraminidase in vivo may well affect the traffic of lymphocytes, with pathological consequences [43]~ ACKNOWLEDGMENTS
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