Download Influenza Virus-specific T Cells Lead to Early Interferon ? in Lungs of

J. gen. Virol. (1989), 70, 975-978. Printedin Great Britain
975
Key words: interferon (MulFN-7)/influenza virus/T cells
Influenza Virus-specific T Cells Lead to Early Interferon ? in Lungs of
Infected Hosts: Development of a Sensitive Radioimmunoassay
By P. M. T A Y L O R , 1. A. M E A G E R 2
AND
B. A. A S K O N A S 1
1Division of Immunology, National Institute for Medical Research, The Ridgeway, Mill Hill,
London N W 7 1AA and 2Division of Immunology, National Institute for Biological Standards and
Control, Blanche Lane, South Mimms, Potters Bar, Hertfordshire EN7 3QG, U.K.
(Accepted 6 December 1988)
SUMMARY
A sensitive immunoradiometric assay for murine interferon ~, (MuIFN-~,) has been
developed and used reproducibly to measure low levels of MulFN-~, in lung lavage
samples from influenza-infected mice. In control infected mice, IFN-~, peaked on day
6, but transfer of virus-specific cytotoxic T cells or T helper cells, which reduced virus
replication in vivo in infected hosts, resulted in an earlier peak on day 4.
Immune interferon (IFN) is the product of activated T cells and its multiple effects on many
cell types are well documented (see for example review by O'Garra et al., 1988). Biological tests
of the antiviral activity of IFN-7 are too insensitive and variable to measure IFN-2: levels in
biological fluids. Development of a highly sensitive specific immunoradiometric assay (IRMA)
that detects 0.01 international units (IU) of IFN-), permitted us to measure the MulFN-~, levels
in lung lavage samples taken from mice during the course of an influenza virus infection.
Furthermore, we find that transfer of cloned influenza-specific T cells which leads to a more
rapid clearance of virus from the lung results in an earlier appearance of MulFN-~, in the
bronchial lavage samples in comparison with intranasal infection per se.
The IRMA we developed for MulFN-y is a solid phase two site sandwich immunoassay based
on the assays for human interferon ~, (HulFN-~) developed by Secher (1981) and Chang et al.
(1984). We used rat anti-MulFN-~ monoclonal antibodies (MAbs) R4-6A2 (Spitalny & Havell,
1984) and AN-18 (Prat et al., 1984). Antibody from ascitic fluid grown in athymic mice (nu/nu)
was precipitated with ammonium sulphate. MAb R4-6A2 was purified by Protein A-Sepharose
4B chromatography, the IgG1 eluted at pH 6 and stored at - 20 °C. R4-6A2 IgG1 was iodinated
with carrier-free ~2sI (Amersham) using the chloramine T method (Greenwood et al., 1963) and
desalted on a Dowex 1- × 8 (C1) column in 0.2~ bovine serum albumin in phosphate-buffered
saline (BSA-PBS) (2 x 107 c.p.m./~tg),
Etched polystyrene beads (Northumbria Biologicals; 6 mm diam.) were left overnight at 4 °C
in AN-18 Ig solution (200 ~tg protein/ml PBS). These beads were then washed four or five times
with 0.5~ BSA-PBS, and stored at 4 °C. Serial dilutions (in 0-5~ BSA-PBS) of MulFN-~
(international standard or laboratory samples; 225 gl/tube) were transferred to Luckham LP4
tubes, after excess binding sites had been blocked with 5 ~ BSA-PBS for 1 h. One AN-18 MAbcoated bead (blotted dry on paper) was added per tube. After 18 h at 4 °C, the beads were washed
three times with 3 ml 0.5~ BSA-PBS. ~zSI-R4-6A2 (2 × 105 c.p.m./225 ~tl 0.5~ BSA-PBS) was
added to the beads for 4 h, followed by three washes with 0.5 ~ BSA-PBS, before counting in an
LKB gamma counter.
The dose-response curve showed linearity over the range of 0.01 to 10 IU/ml using the
international standard for MulFN-~ Gg02-901-533 (Fig. 1); the coefficient of variation was
< 10~ between 0.075 and 9.0 IU/ml. This I R M A has a detection limit of 0-01 IU/ml (100 c.p.m.
bound above non-specific ~25i_Ab binding of 100 to 200 c.p.m./bead). MulFN-~ after treatment
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Fig. 1
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4
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Time post-infection (days)
Fig. 2
Fig. 1. Calibration curves generated in MulFN-specific IRMA by serial dilutions of international
standard MulFN-~ Gg02-901-533 (O), a preparation of MulFN-~, derived from CHO cells (O) and the
same CHO IFN preparation treated at pH 2 for 19 h at 4 °C (A). The ordinate scale is in c.p.m, above
background binding of radioactivity to beads.
Fig. 2. Kinetics of IFN-), release in lungs of influenza-infected mice. BALB/c mice infected i.n. with
A/X31 virus were killed with a lethal dose intraperitoneally of pentobarbitone and 1 ml of PBS was
delivered into the lungs through a small incision in the trachea via 1.2 mm (outer diam.) Portex tubing
with a bevelled tip, attached to a 18 gauge needle and 1 ml syringe. BAL samples were obtained by three
successive insertions and withdrawals of the PBS and cell-free supernatants were stored at - 7 0 °C
(Cannon et al., 1988). IFN-y content of pooled BAL samples (three mice/group) at the times shown was
assayed using the described IRMA. On completion of the BAL procedure, lungs were removed and
homogenized for influenza virus titration in the allantoic cavities of 10-day-old chick embryos (Lin &
Askonas, 1981). Lung virus titre (O); lung lavage IFN-), titre (O).
at p H 2 for 18 h retained 3 to 4 % of its immunochemical activity, a result similar to that of Curry
et al. (1987). The I R M A was specific for MulFN-~, and did not detect H u l F N - 7 , or mouse or
human IFN-~/fl.
We wished to apply this highly sensitive assay to try to assess the kinetics of M u l F N - y release
in bronchial lavage samples from lungs of influenza-infected mice. BALB/c mice were infected
intranasally (i.n.) with A/X31 virus. Fig. 2 shows that lung virus titres peak on days 3 to 6, after
which point there is rapid viral clearance. A sharp peak of MulFN-~, appears on day 6 postinfection, at a time when T cell-mediated cytotoxicity is detected in the lungs of infected mice
(Yap & Ada, 1978). The small amounts of M u l F N - ~ in bronchoalveolar lavage (BAL) samples
(Cannon et al., 1988) are not detected by the conventional biological assays which are not very
sensitive and also suffer from considerable variation between assays (Meager, 1987).
Transfer of virus-specific cytotoxic T (Tc) cell clones into influenza-infected mice does not
prevent infection but speeds up viral clearance and recovery. CD4 ÷ T helper (TH) clones or lines
are much more variable in their effects on viral clearance (Askonas et al., 1988), but CD4 ÷ line
BAE5 is protective. Both cloned Tc and TH ceils have the ability to secrete I F N - ~ on contact
with specific antigen. Tc and TH cell clones or lines produce similar levels of I F N - 7 in vitro
(Table 1). We wished to see whether we could detect earlier release of I F N - 7 into B A L of T cell
recipient mice infected with influenza virus. D a y 4 was selected for sampling, because by day 6
there is a peak of I F N - 7 from the host in response to the virus infection. Table 2 shows that low
but reproducible and therefore significant release of I F N - 7 is detected on day 4 in B A L following
the transfer of Tc or TH lines or clones. Both the cloned Tc and Ta lines are effective in reducing
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Table 1. Antigen-specific IFN-~ production by influenza-specific Tc and Tn clones
c
Specificity
Clone*
BAE5
2A12
2F3
T5/5
T9/13
T8
TH
TH
Tc
Tc
HA
HA
NP
NP
NP
Antigen
J'
Stimulation
~
IFN-7
(IU/ml)t
U.v. A/X31
U.v. A/X31
NP
A/X31-infected P815
A/X31-infected P815
2300
2100
1200
900
2550
* BAE5 is a T Hcell line. T¢ or T H(Taylor & Askonas, 1986; Thomas et al., 1986)were incubated at 5 x 105/ml in
RPMI/10 for 24 h with appropriate antigen stimulation. For Tm this was u.v.-inactivated A/X31 (100 haemagglutination units/m|) or purified influenza nucleoprotein (1 ~tg/ml)and syngeneic, irradiated normal spleen cells as
antigen-presenting cells (2 x 106/ml). For Tc, P815 ceils which had been infected with A/X31 virus for 90 min,
then washed, Were used at 5 x 10S/ml.
~"IFN-7 in the supernatant was assayed by IRMA.
Table 2. IFN-~ in BAL samples following transfer of influenza-specific Tc or Tn into infected
mice
Clone*
Time
post-infection
(days)
Lung virus titre
(logl0/EIDs0)
BAL IFN-~,
(IU/ml/mouse + S.E.M.)f
Tc
T5/5
Control
Tc
T5/5
Control
TH BAE5
Control
TH BAE5
Control
4
4
6
6
4
4
6
6
4.8
5.5
4.0
5.8
4.5
5.5
2.8
5-2
0-75 + 0.21
0.03 + 0.01
ND
ND
2'49 + 1.04
0.02 + 0.01
ND
ND
* Cloned Tc or TH (8 × 106) were transferred intravenously into BALB/c mice 2 h after i.n. infection with
A/X31 virus (Taylor & Askonas, 1986). Four or 6 days later, lungs were lavaged with 1 ml PBS.
5"The IFN-7 content of lung lavage samples from individual mice (four mice/group) was assayed using the
described IRMA.
lung virus titres by day 6, with slight reductions already occurring by day 4 (Table 2). The T H line
produces more I F N - ~ than the Tc clone T5/5 and in fact this is reflected in the amounts of IFN-~,
in B A L samples. The IFN-~ level in B A L samples is low, but reproducible in our experiments.
Thus we observe the a p p e a r a n c e of IFN-~, in lung lavage samples 4 days after influenza
infection following TH or Tc cell transfers which lead to earlier viral clearance, whereas I F N - ?
can only be detected by day 6 in B A L samples from control influenza-infected mice which start
to clear the virus after day 6. There is a discrepancy between the low I F N - ? titres found in B A L
samples compared to the high amounts detected in a culture of 5 x 105 T cells/ml. This is not
surprising since only a low proportion of the transferred cultured T cells reach the sites of
infection because of migration problems (Dailey et al., 1982), and I F N - ~ in vivo has a very short
half life. Furthermore, the volume of the BAL fluid will reflect the large dilution of what is
probably highly localized I F N - ? production by T cells in the lung tissues. Lukacher et al. (1984)
also have shown specifically localized Tc activity that m a y not all be accessible to BAL, which
only samples from superficial areas of the bronchoalveolar lung passages.
Our experiments demonstrate the high sensitivity of the I R M A for assaying low amounts of
M u l F N - ~ in biological fluids to enable a study of kinetics of I F N - ~ release in vivo. The effects we
noted on the reduction of infectious influenza virus by transferred T cells m a y be due to the
direct antiviral activity of IFN-~,, although this is not thought to be a major function of this
particular species of I F N (e.g. Landolfo et al., 1988). Interferons ~ and fl are known to be potent
antiviral agents and I F N - a peaks in the lungs of influenza-infected mice on day 4 (Wyde et al.,
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978
1982). A m o r e likely e x p l a n a t i o n lies w i t h t h e m u l t i p l e i m m u n o r e g u l a t o r y effects o f I F N - y , but,
in c o n j u n c t i o n w i t h t h e effector f u n c t i o n o f T ceils in t h e l u n g s o f i n f l u e n z a - i n f e c t e d m i c e , its
p r e c i s e role r e m a i n s to be e x p l o r e d f u r t h e r .
The influenza-specific TH line BAE5 was selected and generously donated for these experiments by Dr D. B.
Thomas (NIMR). TH clones 2A12 and 2F3 were selected and kindly donated by Mr F. Esquivel (NIMR).
Hybridoma cells producing MAb R4-6A2 were generously provided by Dr E. Havell of the Trudeau Institute,
Saranac Lake, N.Y., U.S.A. and those producing MAb AN-18 by Dr S. Landolfo of the University of Turin, Italy.
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