Download Cytotoxic T Cell Lysis of Target Cells Fused with Liposomes

J. gen. Virol. (1985), 66, 1333-1339.
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1333
Key words: influenza A virus/ C TL/ lysis/ haemagglutinin
Cytotoxic T Cell Lysis of Target Cells Fused with Liposomes Containing
Influenza Virus Haemagglutinin and Neuraminidase
By L O T H A R S T I T Z , 2. R I C H A R D T. C. H U A N G , 3 H A N S
H E N G A R T N E R , 1 R U D O L F R O T T 2 AND R O L F M. Z I N K E R N A G E L ~
l Institute for Pathology, Department of Experimental Pathology, University Hospital, 8091
Zurich, Switzerland, 2Institute for Virology, Justus-Liebig University Giessen, Frankfurterstrasse
107, D-6300 Giessen, Federal Republic of Germany and 3Institute for Molecular Biology and
Biochemistry, Free University, Berlin, Federal Republic of Germany
(Accepted 8 February 1985)
SUMMARY
The lytic activity of secondary cytotoxic lymphocytes against influenza A virus was
tested on cells which had been fused with liposomes containing the haemagglutinin and
the neuraminidase of an avian influenza A virus (fowl plague virus, FPV). Fusion was
obtained solely by the activity of the haemagglutinin and neuraminidase incorporated
into the liposomes, without the need for any additional fusion factor. Highly
reproducible lysis of these FPV-liposome target cells by influenza A-specific cytotoxic
cells was found. In contrast, target cells containing the glycoproteins HN and F of
Newcastle disease virus (NDV) were not lysed. In almost all experiments effector cell
populations capable of lysing target cells also lysed the natural killer cell (NK)-sensitive
cell line YAC-1. However, high NK activity alone was not sufficient to lyse target cells
fused with liposomes containing the viral surface glycoproteins. To our knowledge this
is the first report where after artificial introduction of viral surface components into cell
membranes (either by fusion or by transfection) lysis of target cells was monitored also
for non-specific lysis mediated by NK-like cells. Both the H-2 restriction and the virus
specificity of lysis of FPV-liposome target cells indicate that influenza virus
haemagglutinin and possibly neuraminidase do function as target antigens for
influenza-specific T cells.
Substantial epidemiological and experimental evidence suggests that both T cells and
antibodies play a major role in recovery from and protection against influenza virus infection
(Schulman, 1975). Anti-haemagglutinin (anti-HA) antibodies are of particular importance since
they neutralize the virus; the relevant antigenic sites have been demonstrated elegantly by
Caton et al. (1982) as being the globular HA1 portion of the HA. The biologically important
virus-induced antigen(s) on influenza virus-infected cells recognized by cytotoxic T cells (CTL)
is (are) still largely unknown. Influenza A-immune CTL show cross-reactivity for all influenza A
viruses but sometimes also more restricted subtype specificity (Braciale, 1977a; Doherty et al.,
1977; Effros et al., 1977; Reiss & Schulman, 1980; Zweerink et al., 1977a, b). Common and/or
cross-reactive antigens could easily explain the high extent of cross-reactivity found for primary
or secondary anti-influenza CTL. The importance of the HA as candidate antigen for crossreactive T cells has been de-emphasized (Bennink et al., 1982; Braciale, 1977b; Kees &
Krammer, 1984; Shaw et al., 1981 ; Townsend et al., 1984; Virelizier et al., 1977; Yewdell et al.,
1981) or emphasized, particularly when HA specificity was selected during secondary immune
stimulation (Braciale et at., 1984; Effros et al., 1977 ; Wabuke-Bunoti & Fan, 1983). The present
study demonstrates that HA and neuraminidase (NA) in liposomes fused with target cells can
serve as target antigens for influenza-specific CTL; this antigen-specific T cell activity could be
clearly separated from natural killer cell (NK) reactivities.
0000-6429 © 1985 SGM
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Table 1. Lysis of target cells fused with liposomes containing surface glycoproteins from influenza
A virus (FPIO and of YAC-I*
Specific lysis of target cells
A
Mc57G treated with
A
f
Effector cells
Spleen FPV
FPV
Lip. 1
26
15
10
19
15
7
2
0
1
4
•
FPV
Lip. 3
44
31
21
39
26
17
0
7
2
5
Macrophages uninf.
FPV
71
65
36
92
47
27
5
0
0
0
1
2
2
0
0
Spontaneous release
0
20
I
22
0
16
0
16
0
12
Macrophages FPV
Spleen uninf.
Uninf.
16
10
6
9
6
2
2
3
2
1
YAC-1
33
16
12
43
23
11
3
0
0
0
* Spleen cells from FPV-primed C57BL/6 mice were restimulated in vitro for 5 days with infected or uninfected
syngeneic cells. Mc57G target cells were used either uninfected or FPV-infected or were fused with FPVliposomes. To assess cytolytic activity, best results were obtained from experiments where the fusion was carried
out at 37 °C for 30min in medium (pH 7.2) containing 1~ foetal calf serum (FCS) after preincubation at 4 °C for 15
min. After fusion, cells were incubated at 37 °C for an additional lh (Lip. 1) or 3h (Lip. 3) before they or
uninfected (Uninf.) cells were used as target cells in a cytotoxicityassay. The test was incubated for 4 h at the three
E:T ratios (15:1, 7:1, 3:1).
Virus strains used were h u m a n influenza A strain PR8 (A/PR/8/34; H1N1) and the avian
strain A/FPV/Rostock/34 (fowl plague virus; FPV) (H7N1), the h u m a n influenza B strain Lee
(B/Lee), Newcastle disease virus (NDV: an unrelated paramyxovirus), lymphocytic choriomeningitis virus (LCMV) and vesicular stomatitis virus (VSV) serotype Indiana. BALB/c (H-2d),
C57BL/6 (H-2 b) and C3H/HeJ (H-2 k) mice were purchased from the Institut fiir Zuchthygiene,
Tierspital, Universit~it Ziirich. Mice were immunized by a single intraperitoneal (i.p.) injection
of 50 to 100 haemagglutinating units ( H A U ) in allantoic fluid and were used 4 to 12 weeks later.
Secondary restimulation in vitro was carried out with syngeneic as well as allogeneic stimulators
infected either with the homologous or a heterologous virus (50 to 100 HAU/106 cells). I m m u n e
spleen cells were incubated with acutely infected spleen cells at a responder : stimulator (R : S)
ratio of 1:1 or with acutely infected macrophages at a R :S ratio of 30 : 1. In some experiments,
mice were infected with either LCMV or VSV and their spleens were tested 2 days later. Spleen
cell suspensions were used without further treatment in the cytotoxicity assay.
P815 (H-2d), Mc57G (H-2 b) and L929 (H-2 k) cells were either infected with virus or fused with
liposomes containing viral surface glycoproteins (liposome targets). Target cells were incubated
with approx. 200 H A U of influenza virus per 106 ceils for 1 h at 37 °C in 0. l ml. Thereafter, cells
were washed twice and incubated for another 2 h in medium. During the last hour of incubation
51Cr was added to the cells. CTL activity was tested in conventional SlCr release tests (Bennink
et al., 1982; Doherty et al., 1977) at an effector to target (E :T) cell ratio of 15 : l and twofold
dilutions thereof in 4 to 5 h assays.
Liposomes containing FPV glycoproteins (FPV-liposomes) or glycoproteins from N D V
(NDV-liposomes) were prepared as described previously (Huang et al., 1979). Briefly, virus
particles were solubilized by octylglucoside and viral glycoproteins were obtained from the
supernatant. As shown by gel electrophoresis the supernatant contained exclusively H A and N A
(Huang et al., 1979). This supernatant was mixed with lecithin and cerebroside dissolved in 20~o
octylglucoside at a protein :lipid ratio of 0.09 and stored at - 80 °C. This mixture contained
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Table 2. Specificity of lysis of F P V liposome-containing target cells by various immune
lymphocytes*
% Specific lysis of target cells
L929 treated with
A
Group
I.I
Effector cells
Expt. l : C3H/HeJ FPV-primed restimulated with
Spleen FPV
1.2
Macrophages FPV
1.3
Spleen B/Lee
Spontaneous release
FPV
58
34
18
66
46
24
11
6
4
14
FPV
Lip. 1
36
20
16
23
14
12
16
11
10
12
Uninf.
17
4
1
19
10
3
10
3
2
13
YAC-1
24
17
9
29
24
17
30
15
8
20
P815 treated with
2A
Expt. 2: BALB/c FPV-primed restimulated with
Spleen FPV
2.2
Macrophages FPV
2.3
2 days immune LCMV-primed BALB/c spleen cells
2.4
2 days immune VSV primary BALB/c spleen cells
Spontaneous release
FPV
44
28
13
86
42
31
29
8
5
12
8
3
23
FPV
Lip, l
27
16
14
33
18
4
14
7
0
6
0
0
24
Uninf.
13
4
3
19
11
2
4
4
2
0
0
0
18
YAC-1
15
11
8
13
5
2
80
43
33
71
58
43
18
* Secondarycytotoxiccells were obtained as described in Table 1 and tested in a 4 h assay. E :T ratios were 15 : 1,
7:1 and 3:1. Spleen cells from LCMV (105 p.f.u.) and VSV (107 p.f.u.) acutely infected BALB/c mice were used 2
days after infection at an E :T ratio of 100 :1, 50 : 1 and 25 : 1. Effectors were also tested on the same FPV-liposomecontaining target cells as listed in Table 1. Lysisof these target cell preparations did not exceed the lysis mentioned
in the Table and therefore are not listed.
about 16000 H A U / m l . Prior to fusion, the liposomes were obtained by dialysing the mixture
against medium at 4 °C overnight.
Our experiments demonstrate that surface glycoproteins from influenza A virus fused into
target cells with liposomes can render cells sensitive to lysis by specific i m m u n e T cells from
infected mice after restimulation in vitro (Table 1). However, lysis of the NK-sensitive YAC-1
cell line was significant and comparable to the degree of lysis of FPV-liposome target cells.
Virus-primed spleen cells stimulated in vitro with uninfected spleen or macrophage cells did not
cause lysis of virus-infected, liposome or YAC-1 target cells.
In several experiments we found lysis of FPV-liposome target cells that was higher than that
of the NK-sensitive target cell (Table 2). Additionally, we found examples where high killing of
YAC-1 cells was not accompanied by comparable lysis of FPV-liposome target cells. This
p h e n o m e n o n was observed frequently when effector cells were restimulated in vitro with
stimulator cells infected with another influenza A virus (Expt. 1, group 1.3). Lysis of YAC-1 but
not of FPVqiposome target cells was found when spleen effector cells from mice infected with
LCMV or VSV 2 days previously were tested; these latter effector cells are known to express
high N K activity (Expt. 2, groups 2.3 and 2.4).
The virus specificity of lysis of various target cells was tested on cells infected with FPV, PR8,
B/Lee and on liposome target cells containing glycoproteins either from F P V or from N D V
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T a b l e 3.
Lysis of target cells fused with FPV-glycoprotein- or ND V-glycoprotein-containing
liposomes*
% specific lysis of target cells
x
Mc57G cells incubated with
Group
1
Effector cells
C57BL/6 FPV-primed
restimulated with
C57BL/6 spleen FPV
C57BL/6 spleen PR8
C57BL/6 spleen uninfected
C57BL/6 allantoic fluid
primed restimulated with
C57BL/6 spleen FPV
C57BL/6 PR8-primed
restimulated with
C57BL/6 spleen PR8
C57BL/6 spleen FPV
BALB/c spleen PR8
),
11
BALB/c spleen PR8
Spontaneous release
Uninff
YAC-1
47
25
15
6
16
11
6
2
4
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
25
17
6
0
11
1
1
0
NT
16
11
7
0
NT
NT
0
0
0
0
0
0
0
0
0
0
0
0
2
3
0
0
NT
81
77
60
29
71
69
56
44
8
5
0
0
0
0
0
0
NT
37
25
12
6
31
22
11
9
3
12
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
13
12
8
0
20
17
7
0
29
19
12
8
12
8
2
0
NT
0
0
0
0
50
30
15
6
0
0
0
0
0
0
0
0
5
1
0
3
0
0
0
0
0
0
0
0
0
0
0
0
7
0
0
0
24
10
8
1
11
5
4
0
15
10
4
1
0
0
0
0
0
0
0
0
26
7
11
0
0
12
9
0
1
25
0
0
0
0
8
0
0
0
26
0
0
0
0
0
0
0
0
22
17
7
2
0
8
2
1
0
14
B/Lee
85
72
68
34
63
60
33
12
15
5
0
0
53
43
22
15
49
40
29
18
0
0
0
0
6
0
0
0
NT~
0
0
0
0
0
0
0
0
56
53
40
27
60
64
42
21
10
4
0
0
C57BL/6 allantoic
10
NDV
Lip. 1
PR8
C57BL/6 B/Lee-primed
restimulated with
C57BL/6 spleen B/Lee
BALB/c PR8-primed
restimulated with
BALB/c spleen FPV
FPV
Lip. 1
FPV
NT
0
0
0
0
10
6
0
0
24
0
0
0
0
0
0
0
0
26
NT
NT
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P815
PR8
NT
NT
NT
48
25
14
5
60
30
12
5
24
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1337
(Table 3). Effector cells restimulated with influenza A virus lysed target cells infected with the
same virus slightly better than target cells infected with a different influenza A virus (Table 3,
groups 1 and 5). In contrast, target cells infected with influenza B and uninfected control target
ceils were not lysed by the various anti-influenza A effector cells tested (groups 1 and 5). FPVprimed and restimulated effector cells lysed target cells fused with liposomes containing FPV
glycoprotein to a significant degree but failed to lyse target cells that had been fused with
liposomes containing N D V glycoproteins H N and F. However, in most experiments we
observed considerable lysis of NK-sensitive target cells by the same effector cells (groups 1, 2, 5
and 6). In none of these experiments did we observe lysis of targets fused with N D V components
or cells infected with the heterologous influenza B virus. Effector cells from mice primed with
influenza B virus and restimulated in vitro with the same virus did not lyse any of the influenza Aor NDV-containing targets but lysed B/Lee-infected cells (Table 3, group 8) efficiently; these
effector cells also expressed some N K activity. Additional experiments (not shown) established
clearly that NDV-liposome targets were not lysed by effector cells expressing very high N K
activity against YAC-1 cells.
Spleen ceils from mice immunized with allantoic fluid not containing virus and restimulated
in vitro with virus-infected cells did not cause any lysis of target cells expressing viral antigen
(Table 3, groups 4 and 9). The same was true for virus-primed responder cells incubated in vitro
with allantoic fluid containing no virus (data not shown).
The cytotoxic activity elicited was H-2-restricted and therefore mediated by T cells (Shaw et
al., 1981). Secondary BALB/c (H-2 a) anti-FPV or anti-PR8 effector cells lysed influenza A virusinfected H-2-compatible P815 target cells efficiently (Table 3, groups 10 and 11). The same
effector cells lysed influenza A- or B-infected H-2-incompatible Mc57G cells not at all or only
marginally.
A variety of viral components have been found on cells and discussed as possible target
antigens on the surface of influenza virus-infected cells (Bennink et al., 1982; B raciale, 1977b;
Koszinowski et al., 1980; Kurrle et al., 1979; Townsend et al., 1984; Virelizier et al., 1977;
Yewdell et al., 1981). Recently, Braciale et al. (1984) showed in an elegant study by D N A mediated gene transfer that the expression of the H A on target ceils rendered these cells
susceptible to the lyric activity of subtype-specific and cross-reactive influenza A-specific CTL.
However, in this study [as in the work of Koszinowski et al. (1980) with influenza virus
glycoproteins and of Hale et al. (1981) with VSV surface glycoproteins, both using Sendai virus
as fusion factor] there was no control for non-specific lysis by the NK-like cells which frequently
appear during secondary restimulation in vitro. Involvement of N K lysis has been recently
suggested for Sendai virus glycoprotein-containing target cells (Abidi & Flanagan, 1984).
Taking advantage of the capacity of the described glycoprotein-containing liposomes to fuse
directly into cell membranes (Huang et al., 1980a), we could avoid creating target cells
harbouring viral components other than those from influenza A virus. Only target cells
containing influenza surface components were lysed by influenza immune CTL; N D V
glycoproteins (HN and F) were not able to induce target structures that were recognized. This
also shows that fusion of liposomes with cells does not suffice alone to induce determinants on
target cells that are recognized by influenza virus-specific T cells.
* Spleen cells from virus-primed mice were restimulated with either syngeneic or allogeneic infected, or
uninfected spleen cells. As controls, spleen cells from mice immunized with non-infectious allantoic fluid were
restimulated with virus-infected syngeneic spleen cells. E : T ratios were as described in Table 1. The assay was
terminated after 4 h. Target cells were either uninfected Mc57G, FPV- or PR8-infected cells or cells fused with
FPV-liposomes or NDV-liposomes. These liposomes were from the supernatant and used 1 h after fusion with
tissue culture cells. Secondary C57BL/6 anti-FPV effector cells restimulated with FPV-infected allogeneic (H-2d)
stimulators showed high lysis on Y AC-1 cells (48 ~o at E:T 15:1) but only low or no lysis on all other target cells,
never exceeding 10~ at E :T 15 : 1. These effector cells co-incubated with uninfected syngeneic spleen stimulators
did not cause lysis of any of the target cells. The same was true for secondary C57BL/6 anti-B/Lee effector cells
restimulated with uninfected syngeneic spleen cells, except for low lysis on YAC-1 cells (14~ at E :T 15:1).
t NT, Not tested.
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Table 4. Summary of cell experiments testing lysis of target cells by FPV-induced CTL, after
fusion with liposomes containing viral glycoproteins
Target cells fused
with liposomes containing
glycoproteins from
Influenza A (FPV)
Paramyxovirus (NDV)
No lysis
Lysis accompanied
by low NK
activity
Lysis accompanied
by moderate N K
activity
Lysis accompanied
by high N K
activity
1
4
3
0
2
0
3
1
In more than half of all experiments, good lysis of target cells fused with liposomes containing
viral surface glycoproteins was accompanied by NK-like activity. On the other hand, we clearly
showed that high N K activity induced in secondary cultures in vitro or in mouse spleen cells after
infection with LCMV or VSV in vitro did not on its own cause lysis of liposome target cells to any
great extent. Table 4 shows a summary of all experiments carried out on liposome-fused target
cells with regard to lysis by NK-like ceils.
Since influenza HA does not fuse with cells unless NA is present, we had to include both
glycoproteins in liposomes (Huang et al., 1980b). Therefore our target cells may have been lysed
by either HA- or NA-specific effector cells. In various studies on influenza virus-induced
antigens recognized by T cells, no evidence has been found for NA-specific T subpopulations.
Nevertheless, the possible involvement of NA cannot be excluded in our experiments. The
present and other studies (Braciale et al., 1984; Effros et al., 1977; Wabuke-Bunoti & Fan, 1983)
together certainly support the notion that influenza virus glycoproteins do serve as target
antigens for cytotoxic T cells.
This work was supported by grants Sti 71/1-1 and SFB 47 from the Deutsche Forschungsgemeinschaft, by SNF
3.323-0.82 and USPHSF AI-17285-04.
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