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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 0000-8729 © 1989 SGM Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 03 Aug 2017 09:25:30 976 Short communication 5 I I I I 0 3- I I I I -3~ o6 ~0 / 2 / ~4 -2 z 2. 2~ I I l 0.01 I I I 0.1 1-0 10 IFN titre (IU/ml) Fig. 1 I 100 ~'-~ I I I ~"~---T -'° 2 4 6 8 10 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 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 03 Aug 2017 09:25:30 977 Short communication 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., Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 03 Aug 2017 09:25:30 Short communication 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. REFERENCES ASKONAS,B. A., TAYLOR,P. M & ESQUIVEL,F. (1988). Cytotoxic T cells in influenza infection. Annals of the New York Academy of Sciences 532, 230-237. CANNON, M. J., OPENSHAW, P. J. M. & ASKONAS, B. A. (1988). C y t o t o x i c T-cells clear virus b u t a u g m e n t l u n g p a t h o l o g y in mice infected with respiratory syncytial virus. Journal of Experimental Medicine 168, [ 163-1168. CHANG, T. W., McKINNEY, S., LIU, V., KUNG, P. C., VILCEK, J. & LE, J. (1984). U s e of m o n o c l o n a l a n t i b o d i e s as sensitive and specific probes for biologically active human y-interferon. Proceedings of the National Academy of Sciences, U.S.A. 81, 5219-5222. CURRY, R. C., KIENER, P. A. & SPITALNY, G. L. (1987). A sensitive immunochemical assay for biologically active MulFN- 7. Journal of Immunological Methods 1@4, 137-142. DAILEY, O. M., FATHMAN, G. C., BUTCHER, E. C., PILLEMER, E. & WEISMAN, I. (1982). Abnormal migration of T lymphocyte clones. Journal oflmmunology 128, 2134-2136. GREENWOOD, F. C., HUNTER, W. M. & GLOVER, J. S. (1963). T h e p r e p a r a t i o n of 13 i i . l a b e l l e d h u m a n g r o w t h h o r m o n e of high specific radioactivity. Biochemical Journal 89, 114-123. LANDOLFO, S., GARIGLIO, M., GRIBAUDO, G., JEMMA, C., GIOVARELLI, M. & CAVALLO, G. (1988). Interferon-~ is not an antiviral, but a growth promoting factor for T-lymphocytes. European Journal of Immunology 18, 503-509. LIN, Y. L. & ASKONAS,B. A. (1981). Biological properties of an influenza A virus specific killer T-cell clone. Journalof Experimental Medicine 154, 225-234. LUKACHER, A. E., BRACIALE, V. L. & BRACIALE, T. J. (1984). In vivo effector f u n c t i o n o f influenza virus specific cytotoxic T lymphocyte clones is highly specific. Journal of Experimental Medicine 160, 814-826. MEAGER, A. (1987). Antibodies against interferon - characterisation of interferons and immunoassays. In Lymphokines and Interferons- A PracticalApproach, pp. 105-127. Edited by M. J. Clemens, A. G. Morris & A. J. H. Gearing. Oxford: IRL Press. O'GARRA, A., UMLAND, S., DE FRANCE, T. & CHRISTIANSEN, J. (1988). B cell factors are pleiotropic. ImmunologyToday 9, 45-54. PRAT, M., GRIBAUDO, G., COMOGLIO, P. M., CAVALLO, G. & LANDOLFO, S. (1984). M o n o c l o n a l a n t i b o d i e s a g a i n s t murine y interferon. Proceedings of the National Academy of Sciences, U.S.A. 81, 4515-4519. SECHER, D. S. (1981). Immunoradiometric assay of human leukocyte interferon using monoclonal antibody. Nature, London 29@, 501-503. SI'ITALNY, G. L. & rIAVELL, E. A. (1984). Monoclonal antibody to murine interferon inhibits lymphokine-induced antiviral and macrophage tumoricidal activities. Journal of Experimental Medicine 159, 1560-1565. TAYLOR, P. M. & ASKONAS,B. A. (1986). Influenza nucleoprotein-specific cytotoxic T-cell clones are protective in vivo. Immunology 58, 417-420. THOMAS, D. B., SKEHEL, J. J., MILLS, K. H. G. & GRAHAM, C. M. (1986). Suicide selection of m u r i n e T h e l p e r clones specific for variable regions of the influenza haemagglutinin molecule. European Journal oflmmunotogy 16, 789-793. WYDE, I'. R., WILSON, M. R. & CATE, T. R. (1982). Interferon production by leucocytes infiltrating the lungs of mice during primary influenza virus infection. Infection and lmmunity 38, 1249-1255. YAP, K. L. & ADA, G. L. (1978). Cytotoxic T cells in the lungs of mice infected with influenza A virus. Scandinavian Journal of lmmunology 7, 73-80. (Received 18 October 1988) Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 03 Aug 2017 09:25:30