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Primary pulmonary murine cytotoxic T lymphocyte specificity in respiratory syncytial virus pneumonia
Microbial Pathogenesis 1990 ; 9 : 1 3-18
Primary pulmonary murine cytotoxic T lymphocyte specificity in respiratory syncytial virus pneumonia Renu Gupta,' •* Jonathan W . Yewdell, 1 •t and Jack R . Bennink l,t
Robert A . Olmsted, 2 Peter
L . Collins 2
' The Wistar Institute, 3600 Spruce Street, Philadelphia, Pennsylvania, U .S.A . and 2Laboratory of Infectious Diseases, NIAID, Bethesda, Maryland, U .S.A .
(Received January 25, 1990 ; accepted in revised form April 9, 1990)
Gupta, R . (The Wistar Institute, 3600 Spruce Street, Philadelphia, Pennsylvania, U .S .A.), J . W . Yewdell, R . A . Olmsted, P . L. Collins and J . R . Bennink. Primary pulmonary murine cytotoxic T lymphocyte specificity in respiratory syncytial virus pneumonia . Microbial Pathogenesis 1990 ; 9:13-18 .
Effector cells capable of lysing respiratory syncytial virus (RSV)-infected cells were isolated from the lungs of intranasally infected mice . An examination of their specificity showed that cytolysis was major histocompatibility complex restricted . Using recombinant vaccinia viruses containing cloned RSV genes to infect target cells, BALB/c (H-2 d ) pulmonary effector cells were shown to recognize the fusion protein (F) and to a lesser extent the nucleoprotein (N) . Cells specific for the major glycoprotein (G) or the small hydrophobic protein (1 A) were not observed . Key words:
respiratory syncytial virus ; cytotoxic T lymphocytes ; vaccinia virus ; pneumonia .
Introduction Respiratory syncytial virus (RSV) is a paramyxovirus that causes serious lower respiratory tract illness in infants and young children around the world .' Early attempts at vaccination failed to protect against infection or disease .` In fact, despite the presence of serum-neutralizing and complement-fixing antibodies, morbidity was enhanced upon subsequent RSV infection . Continuing efforts towards the development of a safe and effective vaccine for RSV have met with frustrations -, ' The importance of cell-mediated immunity in preventing or recovering from RSV infections has not been clearly defined . A significant role, however, has been suggested by Cannon et al." who showed that adoptive transfer of T cells from RSV-infected mice into persistently infected immunodeficient mice resulted in reduced pulmonary virus titers . This work follows the demonstration by Bangham et al." of RSV-specific, major histocompatibility complex (MHC)-restricted, cytotoxic T lymphocyte (CTL) precursor cells in the spleens of RSV-infected mice . Bangham et al." and Pemberton et a/." have shown that CTL cultures from BALB/c mice (H-2d ) recognize the viral nucleoprotein (N) and fusion protein (F), but not the major glycoprotein (G) or the ' Present address : The Childrens Hospital of Philadelphia, Civic Center Boulevard, Philadelphia, Pennsylvania, U .S .A . t Present address : Laboratory of Viral Diseases, NIAID, Bethesda, Maryland, U .S .A. 0882-4010/90/070013+06 $03 .00/0
© 1990 Academic Press Limited
14
R . Gupta et al
small hydrophobic protein (1A) . Furthermore, CTL cultures from C57B1/6 (H-2') mice also recognized N but not the G protein (F and 1 A were not tested) . All of the studies published to date have utilized CTL generated by secondary in vitro stimulation . In the present study we first, establish the presence of RSV-specific CTL in lungs of RSV-infected mice, and second, use vaccinia virus recombinants expressing cloned RSV genes to determine their specificity for individual viral gene products .
Results and discussion We initially attempted to secondarily stimulate splenocytes from RSV-infected BALB/c mice (H-2 d ) in vitro using infectious RSV . This resulted in massive proliferation of cells between 2 and 4 days of culture, and cell death by day 5 . Proliferating cells were devoid of virus-specific CTL activity . Stimulation under identical conditions resulted in high levels of CTL activity if UV-irradiated virus was used . Cells from these cultures lysed both the RSV Long strain persistently infected BCH-4 cell line (H-2 d ) and RSV d) A2-infected B8F3 (H-2 targets, but not uninfected B8F3 (H-2 d ) cells (data not shown) . This confirms previous findings of extensive cross-reactive recognition of Long and A2 strains by CTL . 15 The specificity of secondary in vitro cultures was further examined using vaccinia virus recombinants expressing individual cloned RSV genes . For this experiment, cultures were stimulated using paraformaldehyde fixed BCH-4 cells, and the CTL therefore are likely to include only cells that are cross-reactive between A2 and Long strains . Consistent with previous findings, 13,14 strong cross-reactive recognition of RSV-F was observed, as was somewhat weaker recognition of RSV-N and a failure to recognize RSV-G (Table 1) . Extending previous results, 14 we found that cells expressing RSV-1 A were not recognized by these CTL . To examine primary in vivo responses, pooled effector cells obtained from 10 BALB/c mice intranasally infected 6 days previously with RSV were assayed for cytolysis of RSV-infected target cells . Pulmonary effector cells lysed both BCH-4 and RSV-infected B8F3 targets but not uninfected B8F3 (Table 2) . Mediastinal lymph node cells demonstrated a small amount of cytotoxicity against RSV-infected B8F3 cells, but not BCH-4 cells . This cannot be attributed to differences in target cell susceptibility to CTL mediated lysis, since lung effector cells exhibited greater lysis on BCH-4 cells than B8F3 cells . Similar results were obtained in seven other experiments . To determine whether the RSV-specific lysis of primary effector cells was MHC restricted, experiments were performed using pulmonary effector cells from BALB/c (H-2 d ) and CBA/J (H-2 k ) mice . Results showed that BALB/c effector cells lysed RSV-infected B8F3 (H-2 d ) cells but not L929 (H-2 k ) target cells (Table 3) . Reciprocal
Table 1
Cytotoxic T lymphocyte specificity of in vitro restimulated splenocytes Specfic 57 Cr release
Effectors'
RSV-N-VAC
RSV-F-VAC
RSV-G-VAC
RSV-1A-VAC
VACCINIA
1'
2'
11 :1
1 :1
11 :1
1 :1
11 :1
1 :1
11 :1
1 :1
11 :1
1 :1
RSV BCH-4
19
7
71
22
5
0
5
0
0
0
'Splenocytes from two BALB/c mice intranasally infected with RSV were restimulated using paraformaldehyde fixed BCH-4 cells and assayed at the indicated effector-to-target ratios .
Pulmonary CTL specificity for RSV
15
Table 2 Effector cells from mice with respiratory syncytial virus pneumonia % Specific 51 Cr release B8F3 RSV
BCH-4 Effectors'
B8F3
60 :1
30 :1
60 :1
30 :1
60 :1
30 :1
59 0 0
47 0 0
49 10 0
36 5 0
6 0 0
6 0 0
Lungs Lymph nodes Splenocytes
'Pooled effector cells obtained from 10 BALB/c mice infected intranasally with RSV were assayed at the indicated effector-to-target ratios .
Table 3
H-2 restricted lysis of RSV-infected target cells Specific 51 Cr release B8F3 (H-2d ) RSV
L929 (H-2 k )
Uninfected
RSV
Uninfected
Effectors'
25 :1
6 :1
25 :1
6 :1
25 :1
6 :1
CBA/J (H-2 k) BALB/c (H-2° )
-3 61
3 35
3 10
0 -1
38 7
17 3
25 :1 0 3
6 :1 1 -4
'Pooled effector cells obtained from the lungs of 10 RSV-infected CBA or BALB/c mice were assayed at the indicated effector-to-target ratios .
Table 4
Specificity of pulmonary anti-RSV cytotoxic T lymphocytes Specfic 51 Cr release
Effectors' BALB/c lung
RSV-N-VAC
RSV-F-VAC
RSV-G-VAC
RSV-IA-VAC
VACCINIA
25 :1
6 :1
25 :1
6 :1
25 :1
6 :1
25 :1
6 :1
25 :1
6 :1
14
10
30
20
3
2
1
1
0
0
'Pooled effector cells obtained from the lungs of 10 RSV-infected mice were assayed at the indicated effector-to-target ratios .
results were obtained with effector cells obtained from CBA/J lungs . Similar results were obtained in three other experiments . Recombinant vaccinia viruses expressing individual cloned RSV genes were then used to examine which RSV gene products were recognized by BALB/c pulmonary effector cells (Table 4) . Cells expressing F, and to a lesser extent N, were specifically lysed (Table 4) . RSV-1A-VAC- and RSV-G-VAC-infected targets were not lysed . Similar experiments were obtained in seven other experiments . Further experiments confirmed the ability of RSV-F-VAC to immunize mice for secondary in vitro RSV restimulation (data not shown) ." In summary, we have extended the previous studies of RSV-specific CTL by demonstrating that following intranasal infection with RSV, the great bulk of the active RSV-specific CTL are in the lungs . We . further show that the specificities of RSVspecific CTL for individual viral gene products obtained with secondarily stimulated splenocytes are representative of the CTL actually present in the lungs of infected
R . Gupta et al.
16
animals . It will be important in future studies to determine the relative contribution of RSV-specific CTL to recovery from infection versus immune-mediated pathology .
Materials and methods Mice. Male BALB/c (H-2 d ) and CBA/J mice (5-8 weeks old) were purchased from The Jackson Laboratory, Bar Harbour, Maine . Cell lines. P815 cells, a DBA/2 (H-2 d ) mastocytoma cell line, were grown in RPMI 1640 medium supplemented with 7 .5% fetal bovine serum and penicillin/streptomycin (10 4 U/I) . L929 cells, a C3H (H-2k ) connective tissue cell line, were obtained from the American Type Culture Collection, Rockville, Maryland (ATCC-CCL1) . B8F3, a BALB/c (H-2 d ) cell line, and BCH-4, a cell line derived from B8F3 persistently infected with respiratory syncytial virus (RSV) Long strain, were kindly provided by Dr B . Fernie, Georgetown, University, Rockville, Maryland ." L929, BCH-4, and B8F3 cell lines were grown in Iscoves modified Dulbeccos medium (IMDM) supplemented with 7 .5% fetal bovine serum, glutamine (20 mmol/I), sodium bicarbonate (3 .024 g/I) and penicillin/streptomycin (10 4 U/I) (IMDM+) . Viruses . RSV A2 strain was grown in HEp-2 cells in IMDM supplemented with 3% fetal bovine serum, glutamine (20 mmol/I), sodium bicarbonate (3 .024 g/I) and penicillin/streptomycin (10 4 U/I) . 17 Cell monolayers were infected with RSV at a 0 .1 multiplicity of infection (moi) for 2 h at 37°C, incubated at 33°C for 72 h, and the supernatant harvested and stored at -70°C . Virus titers were measured by indirect immunoperoxidase assay on HEp-2 cells infected for 3 days in 96-well microtiter plates as described previously 78 using anti-RSV monoclonal antibodies generously given by Dr B . Fernie, Georgetown University, Rockville, Maryland .' 9,20 All RSV titers were 1-2x 10' syncytia forming units (sfu) per ml . UV-inactivated virus was prepared by exposing 1 .0 ml of stock virus in a 60x15 mm Petri dish to a 4 Watt UV lamp at a distance of 15 cm for 5 min . Recombinant vaccinia viruses [1-3x10 $ plaque forming units (pfu) per ml] expressing the RSV fusion protein (RSV-F-VAC), glycoprotein G (RSV-G-VAC), nucleoprotein (RSV-NVAC), and small hydrophobic protein (RSV-1 A-VAC) were constructed and grown as previously described . 21-23 Preparation of target cells. B8F3 or L929 cell suspensions to be used as infected target cells were incubated at 10' cells per ml in PBS with virus at a moi of 10 for 1 h . The infected cells were then pelleted, resuspended in IMDM+ and incubated for 18 h at 37°C . For labeling, 10' B8F3, L929, or BCH-4 cells were pelleted and incubated with 250 µCi Na 51 CrO, at a concentration of 10 mCi/ml for 1 h . Labeled cells were washed twice and placed into wells of round-bottomed microtiter plates at 104 cells per well . Preparation of effector cells. Primary in vitro effector cells were prepared from mice anesthetized with metophane and infected intranasally with 2 .5x103 sfu of RSV A2 in a 50 pl volume . Mice were inoculated i .v . with 1 x10' pfu of recombinant vaccinia viruses . Cell suspensions were obtained from the lungs of intranasally infected mice as previously described . 24 Cells were washed twice in PBS and resuspended in IMDM+ . Secondary in vitro effector cells were prepared by co-culturing 6x106 stimulator cells with 16x 106 responder splenocytes from mice immunized 2-8 weeks earlier with RSV or recombinant vaccinia virus . Stimulator cells used in these cultures were either BCH-4 cells fixed with 3% parafomaldehyde or autologous spleen cells treated with recombinant vaccina virus (10 pfu/cell) or UV-inactivated RSV (10 pfu/cell) . The co-culturing was for 5 days at 37°C . Cytotoxicity assay. Effector cells were placed into triplicate microtiter wells (100 ul) at appropriate dilutions followed by 10 4 target cells in 100 pl of IMDM . After 4 h at 37°C, supernatants were removed and the concentration of released 51 Cr determined by gamma counting . The percentage of 61 Cr was calculated as follows : . cpm-spont. cpm % specific 51 Cr release = exp x 100 total cpm-spont . cpm
Pulmonary CTL specificity for RSV
17
Spontaneous release (spont . cpm) and total release (total cpm) were determined from target cells incubated with medium alone or 5% Triton X100, respectively . All experimental values (exp . cpm) represent the average of triplicates ; all data points had a standard error of the mean less than 4% specific release .
This work was supported by grants Al 14162, 5-T32A107278 and 5-2891 from the National Institutes of Health, Bethesda, Maryland .
References 1 . McIntosh K, Chanock RM . Respiratory syncytial virus . In : Fields BN, ed . Virology . New York : Raven, 1985;1285-304 . 2 . Chin J, Magoffi RL, Shearer LA, Schieble JH, Lennette EH . Field evaluation of a respiratory syncytial virus vaccine and a trivalent parainfluenza virus vaccine in a pediatric population . Am J Epidemiol 1969; 89 : 449-63 . 3 . Fulginiti VA, Eller JJ, Sieber OF, Joyner JW, Minamitani M, Meiklejohn G . Respiratory virus immunization . I . A field trial of two inactivated respiratory virus vaccines ; an aqueous trivalent parainfluenza virus vaccine and an alum-precipitated respiratory syncytial virus vaccine . Am J Epidemiol 1969 ; 89 : 435-48 . 4 . Kapikian AZ, Mitchell RH, Chanock RM, Shvedoff RA, Stewart CE . An epidemiologic study of altered clinical reactivity to respiratory syncytial (RS) virus infection in children previously vaccinated with an inactivated RS vaccine . Am J Epidemiol 1969 ; 89 : 405-21 . 5 . Kim HW, Canchola JG, Brandt CD et al. Respiratory syncytial virus disease in infants despite prior administration of antigenic inactivated vaccine . Am J Epidemiol 1969 ; 89 : 422-34. 6 . Belshe RB, Van Voris LP, Mufson MA . Parenteral administration of live respiratory syncytial virus vaccine . Results of a field trial . J Infect Dis 1982 ; 145: 311-19 . 7 . Buynak EB, Weibel RE, McLean AA, Hilleman MR . Live respiratory syncytial virus vaccine administered parenterally . Proc Soc Exp Biol Med 1978 ; 157 : 636-42 . 8 . Kim HW, Arrobio JO, Brandt CD et al. Safety and antigenicity of temperature sensitive (TS) mutant respiratory syncytial virus (RSV) in infants and children . Pediatrics 1973; 52 : 56-63 . 9 . Wright PF, Belshe RB, Kim HW, Van Voris LP, Chanock RM . Administration of a highly attenuated live respiratory syncytial virus vaccine to adults and children . Infect Immun 1982 ; 37 : 397-400 . 10 . Wright PF, Mills J, Chanock RM . Evaluation of a temperature sensitive mutant of respiratory syncytial virus in adults . J Infect Dis 1971 ; 124 : 505-11 . 11 . Cannon MJ, Stott EJ, Taylor G, Askonas BA . Clearance of persistent respiratory syncytial virus infections in immunodeficient mice following transfer of primed T cells . Immunology 1987 ; 62 :133-8. 12 . Bangham CRM, Cannon MJ, Karzon DT, Askonas BA . Cytotoxic T-cell response to respiratory syncytial virus in mice . J Virol 1985 ; 56: 55-9 . 13 . Bangham CRM, Openshaw PJM, Ball LA, King AMQ, Wertz GW, Askonas BA. Human and murine cytotoxic T cells specific to respiratory syncytial virus recognize the viral nucleoprotein (N), but not the major glycoprotein (G), expressed by vaccinia virus recombinants . J Immunol 1986 ; 137 : 3973-7 . 14 . Pemberton RM, Cannon MJ, Openshaw PJM, Ball LA, Wertz GW, Askonas BA. Cytotoxic T cell specificity for respiratory syncytial virus proteins : fusion protein is an important target antigen . J Gen Virol 1987 ; 68 : 2177-82 . 15 . Bangham CRM, Askonas BA. Murine cytotoxic T cells specific to respiratory syncytial virus recognize different antigenic subtypes of the virus . J Gen Virol 1986 ; 67 : 623-9. 16 . Fernie BF, Ford EC, Gerin JL . The development of BALB/c cells persistently infected with respiratory syncytial virus : presence of ribonucleoprotein on the cell surface . Proc Soc Exp Biol Med 1981 ; 167 : 83-6 . 17 . Huang YT, Wertz GW . The genome of respiratory syncytial virus is a negative-stranded RNA that codes at least seven mRNA species. J Virol 1982 ; 43:150-7 . 18 . Yewdell JW, Bennink JR, Mackett M, Lefrancois L, Lyles DS, Moss B . Recognition of cloned vesicular stomatitis virus internal and external gene products by cytotoxic T lymphocytes . J Exp Med 1986 ; 163 : 1529-38 . 19 . Cote PJ Jr, Fernie BF, Ford EC, Shih JW, Gerin JL . Monoclonal antibodies to respiratory syncytial virus : detection of virus neutralization and other antigen antibody systems using infected human and murine cells . J Virol Methods 1981 ; 3 :137-47 . 20 . Fernie BF, Cote PJ Jr, Gerin JL . Classification of hybridomas to respiratory syncytial virus glycoproteins . Proc Soc Exp Biol Med 1982; 171 : 226-71 . 21 . Elango N, Prince GA, Murphy BR, Venkatesan S, Chanock RM, Moss B . Resistance to human respiratory syncytial virus (RSV) infection induced by immunization of cotton rats with a recombinant vaccinia virus expressing the RSV G . glycoprotein . Proc Natl Acad Sci USA 1986; 83 : 1906-10. 22 . King AM, Stott EJ, Langer SJ, Young KK, Ball LA, Wertz GW . Recombinant vaccinia viruses carrying the N gene of human respiratory syncytial virus : studies of gene expression in cell culture and immune response in mice . J Virol 1987 ; 61 : 2885-90.
18
R . Gupta et al.
23 . Olmsted RA, Elango N, Prince GA et a/. Expression of the F glycoprotein of respiratory syncytial virus by a recombinant vaccinia virus : Comparison of the individual contributions of the F and G glycoproteins to host immunity . Proc Natl Acad Sci USA 1986 ; 83 : 7462-6 . 24 . Bennink J, Effros RB, Doherty PC . Influenza pneumonia : early appearance of cross-reactive T-cells in lungs of mice primed with heterologous type A virus . Immunol 1978; 35 : 503-10 .
Primary pulmonary murine cytotoxic T lymphocyte specificity in respiratory syncytial virus pneumonia Renu Gupta,' •* Jonathan W . Yewdell, 1 •t and Jack R . Bennink l,t
Robert A . Olmsted, 2 Peter
L . Collins 2
' The Wistar Institute, 3600 Spruce Street, Philadelphia, Pennsylvania, U .S.A . and 2Laboratory of Infectious Diseases, NIAID, Bethesda, Maryland, U .S.A .
(Received January 25, 1990 ; accepted in revised form April 9, 1990)
Gupta, R . (The Wistar Institute, 3600 Spruce Street, Philadelphia, Pennsylvania, U .S .A.), J . W . Yewdell, R . A . Olmsted, P . L. Collins and J . R . Bennink. Primary pulmonary murine cytotoxic T lymphocyte specificity in respiratory syncytial virus pneumonia . Microbial Pathogenesis 1990 ; 9:13-18 .
Effector cells capable of lysing respiratory syncytial virus (RSV)-infected cells were isolated from the lungs of intranasally infected mice . An examination of their specificity showed that cytolysis was major histocompatibility complex restricted . Using recombinant vaccinia viruses containing cloned RSV genes to infect target cells, BALB/c (H-2 d ) pulmonary effector cells were shown to recognize the fusion protein (F) and to a lesser extent the nucleoprotein (N) . Cells specific for the major glycoprotein (G) or the small hydrophobic protein (1 A) were not observed . Key words:
respiratory syncytial virus ; cytotoxic T lymphocytes ; vaccinia virus ; pneumonia .
Introduction Respiratory syncytial virus (RSV) is a paramyxovirus that causes serious lower respiratory tract illness in infants and young children around the world .' Early attempts at vaccination failed to protect against infection or disease .` In fact, despite the presence of serum-neutralizing and complement-fixing antibodies, morbidity was enhanced upon subsequent RSV infection . Continuing efforts towards the development of a safe and effective vaccine for RSV have met with frustrations -, ' The importance of cell-mediated immunity in preventing or recovering from RSV infections has not been clearly defined . A significant role, however, has been suggested by Cannon et al." who showed that adoptive transfer of T cells from RSV-infected mice into persistently infected immunodeficient mice resulted in reduced pulmonary virus titers . This work follows the demonstration by Bangham et al." of RSV-specific, major histocompatibility complex (MHC)-restricted, cytotoxic T lymphocyte (CTL) precursor cells in the spleens of RSV-infected mice . Bangham et al." and Pemberton et a/." have shown that CTL cultures from BALB/c mice (H-2d ) recognize the viral nucleoprotein (N) and fusion protein (F), but not the major glycoprotein (G) or the ' Present address : The Childrens Hospital of Philadelphia, Civic Center Boulevard, Philadelphia, Pennsylvania, U .S .A . t Present address : Laboratory of Viral Diseases, NIAID, Bethesda, Maryland, U .S .A. 0882-4010/90/070013+06 $03 .00/0
© 1990 Academic Press Limited
14
R . Gupta et al
small hydrophobic protein (1A) . Furthermore, CTL cultures from C57B1/6 (H-2') mice also recognized N but not the G protein (F and 1 A were not tested) . All of the studies published to date have utilized CTL generated by secondary in vitro stimulation . In the present study we first, establish the presence of RSV-specific CTL in lungs of RSV-infected mice, and second, use vaccinia virus recombinants expressing cloned RSV genes to determine their specificity for individual viral gene products .
Results and discussion We initially attempted to secondarily stimulate splenocytes from RSV-infected BALB/c mice (H-2 d ) in vitro using infectious RSV . This resulted in massive proliferation of cells between 2 and 4 days of culture, and cell death by day 5 . Proliferating cells were devoid of virus-specific CTL activity . Stimulation under identical conditions resulted in high levels of CTL activity if UV-irradiated virus was used . Cells from these cultures lysed both the RSV Long strain persistently infected BCH-4 cell line (H-2 d ) and RSV d) A2-infected B8F3 (H-2 targets, but not uninfected B8F3 (H-2 d ) cells (data not shown) . This confirms previous findings of extensive cross-reactive recognition of Long and A2 strains by CTL . 15 The specificity of secondary in vitro cultures was further examined using vaccinia virus recombinants expressing individual cloned RSV genes . For this experiment, cultures were stimulated using paraformaldehyde fixed BCH-4 cells, and the CTL therefore are likely to include only cells that are cross-reactive between A2 and Long strains . Consistent with previous findings, 13,14 strong cross-reactive recognition of RSV-F was observed, as was somewhat weaker recognition of RSV-N and a failure to recognize RSV-G (Table 1) . Extending previous results, 14 we found that cells expressing RSV-1 A were not recognized by these CTL . To examine primary in vivo responses, pooled effector cells obtained from 10 BALB/c mice intranasally infected 6 days previously with RSV were assayed for cytolysis of RSV-infected target cells . Pulmonary effector cells lysed both BCH-4 and RSV-infected B8F3 targets but not uninfected B8F3 (Table 2) . Mediastinal lymph node cells demonstrated a small amount of cytotoxicity against RSV-infected B8F3 cells, but not BCH-4 cells . This cannot be attributed to differences in target cell susceptibility to CTL mediated lysis, since lung effector cells exhibited greater lysis on BCH-4 cells than B8F3 cells . Similar results were obtained in seven other experiments . To determine whether the RSV-specific lysis of primary effector cells was MHC restricted, experiments were performed using pulmonary effector cells from BALB/c (H-2 d ) and CBA/J (H-2 k ) mice . Results showed that BALB/c effector cells lysed RSV-infected B8F3 (H-2 d ) cells but not L929 (H-2 k ) target cells (Table 3) . Reciprocal
Table 1
Cytotoxic T lymphocyte specificity of in vitro restimulated splenocytes Specfic 57 Cr release
Effectors'
RSV-N-VAC
RSV-F-VAC
RSV-G-VAC
RSV-1A-VAC
VACCINIA
1'
2'
11 :1
1 :1
11 :1
1 :1
11 :1
1 :1
11 :1
1 :1
11 :1
1 :1
RSV BCH-4
19
7
71
22
5
0
5
0
0
0
'Splenocytes from two BALB/c mice intranasally infected with RSV were restimulated using paraformaldehyde fixed BCH-4 cells and assayed at the indicated effector-to-target ratios .
Pulmonary CTL specificity for RSV
15
Table 2 Effector cells from mice with respiratory syncytial virus pneumonia % Specific 51 Cr release B8F3 RSV
BCH-4 Effectors'
B8F3
60 :1
30 :1
60 :1
30 :1
60 :1
30 :1
59 0 0
47 0 0
49 10 0
36 5 0
6 0 0
6 0 0
Lungs Lymph nodes Splenocytes
'Pooled effector cells obtained from 10 BALB/c mice infected intranasally with RSV were assayed at the indicated effector-to-target ratios .
Table 3
H-2 restricted lysis of RSV-infected target cells Specific 51 Cr release B8F3 (H-2d ) RSV
L929 (H-2 k )
Uninfected
RSV
Uninfected
Effectors'
25 :1
6 :1
25 :1
6 :1
25 :1
6 :1
CBA/J (H-2 k) BALB/c (H-2° )
-3 61
3 35
3 10
0 -1
38 7
17 3
25 :1 0 3
6 :1 1 -4
'Pooled effector cells obtained from the lungs of 10 RSV-infected CBA or BALB/c mice were assayed at the indicated effector-to-target ratios .
Table 4
Specificity of pulmonary anti-RSV cytotoxic T lymphocytes Specfic 51 Cr release
Effectors' BALB/c lung
RSV-N-VAC
RSV-F-VAC
RSV-G-VAC
RSV-IA-VAC
VACCINIA
25 :1
6 :1
25 :1
6 :1
25 :1
6 :1
25 :1
6 :1
25 :1
6 :1
14
10
30
20
3
2
1
1
0
0
'Pooled effector cells obtained from the lungs of 10 RSV-infected mice were assayed at the indicated effector-to-target ratios .
results were obtained with effector cells obtained from CBA/J lungs . Similar results were obtained in three other experiments . Recombinant vaccinia viruses expressing individual cloned RSV genes were then used to examine which RSV gene products were recognized by BALB/c pulmonary effector cells (Table 4) . Cells expressing F, and to a lesser extent N, were specifically lysed (Table 4) . RSV-1A-VAC- and RSV-G-VAC-infected targets were not lysed . Similar experiments were obtained in seven other experiments . Further experiments confirmed the ability of RSV-F-VAC to immunize mice for secondary in vitro RSV restimulation (data not shown) ." In summary, we have extended the previous studies of RSV-specific CTL by demonstrating that following intranasal infection with RSV, the great bulk of the active RSV-specific CTL are in the lungs . We . further show that the specificities of RSVspecific CTL for individual viral gene products obtained with secondarily stimulated splenocytes are representative of the CTL actually present in the lungs of infected
R . Gupta et al.
16
animals . It will be important in future studies to determine the relative contribution of RSV-specific CTL to recovery from infection versus immune-mediated pathology .
Materials and methods Mice. Male BALB/c (H-2 d ) and CBA/J mice (5-8 weeks old) were purchased from The Jackson Laboratory, Bar Harbour, Maine . Cell lines. P815 cells, a DBA/2 (H-2 d ) mastocytoma cell line, were grown in RPMI 1640 medium supplemented with 7 .5% fetal bovine serum and penicillin/streptomycin (10 4 U/I) . L929 cells, a C3H (H-2k ) connective tissue cell line, were obtained from the American Type Culture Collection, Rockville, Maryland (ATCC-CCL1) . B8F3, a BALB/c (H-2 d ) cell line, and BCH-4, a cell line derived from B8F3 persistently infected with respiratory syncytial virus (RSV) Long strain, were kindly provided by Dr B . Fernie, Georgetown, University, Rockville, Maryland ." L929, BCH-4, and B8F3 cell lines were grown in Iscoves modified Dulbeccos medium (IMDM) supplemented with 7 .5% fetal bovine serum, glutamine (20 mmol/I), sodium bicarbonate (3 .024 g/I) and penicillin/streptomycin (10 4 U/I) (IMDM+) . Viruses . RSV A2 strain was grown in HEp-2 cells in IMDM supplemented with 3% fetal bovine serum, glutamine (20 mmol/I), sodium bicarbonate (3 .024 g/I) and penicillin/streptomycin (10 4 U/I) . 17 Cell monolayers were infected with RSV at a 0 .1 multiplicity of infection (moi) for 2 h at 37°C, incubated at 33°C for 72 h, and the supernatant harvested and stored at -70°C . Virus titers were measured by indirect immunoperoxidase assay on HEp-2 cells infected for 3 days in 96-well microtiter plates as described previously 78 using anti-RSV monoclonal antibodies generously given by Dr B . Fernie, Georgetown University, Rockville, Maryland .' 9,20 All RSV titers were 1-2x 10' syncytia forming units (sfu) per ml . UV-inactivated virus was prepared by exposing 1 .0 ml of stock virus in a 60x15 mm Petri dish to a 4 Watt UV lamp at a distance of 15 cm for 5 min . Recombinant vaccinia viruses [1-3x10 $ plaque forming units (pfu) per ml] expressing the RSV fusion protein (RSV-F-VAC), glycoprotein G (RSV-G-VAC), nucleoprotein (RSV-NVAC), and small hydrophobic protein (RSV-1 A-VAC) were constructed and grown as previously described . 21-23 Preparation of target cells. B8F3 or L929 cell suspensions to be used as infected target cells were incubated at 10' cells per ml in PBS with virus at a moi of 10 for 1 h . The infected cells were then pelleted, resuspended in IMDM+ and incubated for 18 h at 37°C . For labeling, 10' B8F3, L929, or BCH-4 cells were pelleted and incubated with 250 µCi Na 51 CrO, at a concentration of 10 mCi/ml for 1 h . Labeled cells were washed twice and placed into wells of round-bottomed microtiter plates at 104 cells per well . Preparation of effector cells. Primary in vitro effector cells were prepared from mice anesthetized with metophane and infected intranasally with 2 .5x103 sfu of RSV A2 in a 50 pl volume . Mice were inoculated i .v . with 1 x10' pfu of recombinant vaccinia viruses . Cell suspensions were obtained from the lungs of intranasally infected mice as previously described . 24 Cells were washed twice in PBS and resuspended in IMDM+ . Secondary in vitro effector cells were prepared by co-culturing 6x106 stimulator cells with 16x 106 responder splenocytes from mice immunized 2-8 weeks earlier with RSV or recombinant vaccinia virus . Stimulator cells used in these cultures were either BCH-4 cells fixed with 3% parafomaldehyde or autologous spleen cells treated with recombinant vaccina virus (10 pfu/cell) or UV-inactivated RSV (10 pfu/cell) . The co-culturing was for 5 days at 37°C . Cytotoxicity assay. Effector cells were placed into triplicate microtiter wells (100 ul) at appropriate dilutions followed by 10 4 target cells in 100 pl of IMDM . After 4 h at 37°C, supernatants were removed and the concentration of released 51 Cr determined by gamma counting . The percentage of 61 Cr was calculated as follows : . cpm-spont. cpm % specific 51 Cr release = exp x 100 total cpm-spont . cpm
Pulmonary CTL specificity for RSV
17
Spontaneous release (spont . cpm) and total release (total cpm) were determined from target cells incubated with medium alone or 5% Triton X100, respectively . All experimental values (exp . cpm) represent the average of triplicates ; all data points had a standard error of the mean less than 4% specific release .
This work was supported by grants Al 14162, 5-T32A107278 and 5-2891 from the National Institutes of Health, Bethesda, Maryland .
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