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Specific recognition of influenza virus polymerase protein (PB1) by a murine cytotoxic T-Cell clone
VIROLOGY
160,278-280
(1987)
Specific Recognition
of Influenza
JUDY M. BASTIN,’ Nuffield
Department
of Clinical
Virus Polymerase
Protein
ALAIN R. M. TOWNSEND,
Medicine, Received
John Radcliffe February
Hospital!
13, 1987;
(PBl)
by a Murine
Cytotoxic
AND ANDREW J. MCMICHAEL Headington,
accepted
April
Oxford
OX3 9DU,
Copyright All rights
$3.00
0 1987 by Academic Press. Inc. of reproduction in any form reserved.
Kingdom
influenza virus. virus expressing
by repeated antigenic stimulation in the same conditioned medium. Lytic clones were tested in a standard 6-hr 5’Cr release assay using L cells infected with recombinant vaccinia virus containing influenza cDNA sequences from A/PW8/34 virus (at a multiplicity of infection of 10: 1) and with whole influenza virus. The recombinant vaccinia viruses used were provided by Dr. B. Moss (NIH) and Dr. G. Smith (Cambridge) and contained cDNA corresponding to influenza A/PR/8/34 virus RNA segment 1 (PB2-VAC), segment 2 (PBl -VAC), segment 3 (PA-VAC), segment 4 (H 1-VAC), segment 5 (NP-VAC), segment 7 (M-VAC), and segment 8 (NSlVAC). In addition H2-VAC containing cDNA of segment 4 of AIJAP/305/1957 influenza virus was used. Clone 2-l 6-4 was shown to lyse L cells infected with either influenza A virus A/PR/8/34 (Hl N 1) or A/NT/601 68 (H3N2). However, it did not lyse target cells transfected with NP cDNA of influenza A/NT/60/68 virus. This implied that the cross-reactive clone 2-l 6-4 may recognise some component of influenza virus other than nucleoprotein. It was therefore tested on L cells infected with a series of recombinant vaccinia viruses which each express one of the remaining influenza proteins. Table 1 shows the recognition by clone 2-l 64 (99.7% Ly2 positive by flow cytometric analysis) of L-cell targets infected with PBl-VAC and A/NT/60/68 virus but not of cells infected with vaccinia without influenza cDNA inserts. In addition it shows that this clone did not recognise targets infected with recombinant vaccinia expressing the following influenza proteins: PB2, PA, NP, H2, Hl, NSl, and matrix. Table 1 also shows the recognition by a lo-week-old CBA influenza-specific CTL line 3-9-4, of both PBl and NP inserts but not the others tested. In order to show that the PBl vaccinia-infected targets did not become susceptible to nonspecific killer activity that might be in the T-cell cultures, a C57 Db restricted CTL clone, F5, specific for 1968 NP (4) was used as a negative control. This population of cells did not lyse either PBl
’ To whom requests for reprints should be addressed. * Abbreviations used: CTL, cytotoxic T lymphocytes; PBl , polymerase protein 1 (basic); PB2, polymerase protein 2 (basic); PA, polymerase protein (acidic); Hl, haemagglutinin type 1; H2, haemagglutinin type 2; M, matrix protein; NP, nucleoprotein; NSl, nonstructural protein; VAC vaccinia. 0042-6822187
United
3, 1987
Cytotoxic T-cell clones were raised in CBA mice that recognised both A/X31 and A/JAP/305/1957 Here, we describe one CTL clone that recognises target cells infected with a recombinant vaccinia influenza PBl . a3 1997 Academic press, hc.
Major histocompatibility complex restricted virus specific cytotoxic T lymphocytes (CTL)’ are a prominent feature of host immune responses to many viruses, where they appear to play a role limiting viral replication and dissemination (1, 2). The majority of murine CTL recognises a cross-reactive determinant shared by influenza A but not B viruses, minor populations can distinguish between influenza A subtypes (3). The viral antigens recognised by cross-reactive anti-influenza virus CTL remained largely undefined until recently when it became possible to insert defined viral genes into target cells by DNA transfection (4, 5) and also by the use of recombinant vaccinia virus (6, 7). Use of these methods and more recently use of synthesised peptides of viral proteins has shown that a major population of cross-reactive CTL recognises influenza A virus nucleoprotein (4, 6, 8). Here we describe a murine CTL clone that can recognise target cells that express another internal protein of influenza virus, i.e., polymerase protein PBl . All the methods used to derive and test influenza Aspecific murine CTL have been described in detail elsewhere (8). Briefly, polyclonal cytotoxic lines were established by repeated stimulation in vitro of spleen cells from intraperitoneally primed (A/JAP/305/1957 influenza virus) donor CBA (H2K) mice, with irradiated influenza A/X3 1 or A/PRf8/34 virus-infected syngeneic spleen cells. After two stimulations, T-cell growth factors in the form of supernatant from concanavalin Apulsed rat spleen cells were added to a final concentration of 20% (v/v). In order to raise NP specific clones, CTL were cloned by limiting dilution onto an influenza A/X31 -infected spleen cell feeder layer and maintained
T-Cell Clone
278
SHORT
COMMUNICATIONS TABLE
CV~OTOXICITY Effector KIT ratio
279
1
DATA ON CLONE 2/l 6/4 AND LINE 3/9/4
(PERCENTAGE SPECIFIC “Cr
RELEASE)~
A/NT60/68
V
PBl
PB2
PA
NP
H2
Hl
NSl
Matrix
Uninfected cells
2/l 614 CTL clone
10 5 2.5
29.5 23.1 15.9
2.8 0.0 0.0
39.4 26.8 23.2
4.1 3.2 0.0
1.6 1.7 1.3
6.9 8.0 3.6
0.6 0.1 0.0
8.4 3.3 7.5
4.4 0.8 2.3
5.3 4.0 4.0
NT NT NT
3/g/4 CTL line
10 5 2.5
50.9 41.7 16.0
3.9 0.0 0.0
43.5 23.5 10.1
9.2 4.1 0.0
6.3 4.6 5.2
97.6 50.3 26.3
0.0 0.0 0.0
7.4 5.2 4.5
2.7 2.4 0.9
8.8 0.0 0.3
NT NT NT
5.0 4.5 5.0
8.3 5.6 3.5
2.7 2.4 0.8
3.4 2.7 2.4
19.6 12.3 7.4
NT NT NT
11.3 6.4 3.4
8.1 5.8 4.5
NT NT NT
4.5 3.2 2.3
in medium
alone.
A/PFU8/34 6-Day CBA polyclonal line
15 7.5 3.75
36.4 31.1 18.4
Nore. Each point was measured in duplicate against CTL ranged between 9 and 29% of the Triton release. B Percentage
specific
5’Cr release
was calculated
as
quadriplicate (release
controls
in presenceof (Briton
vaccinia-infected or nonrecombinant vaccinia-infected (0.5% specific release) targets, but did lyse ANT60/68 infected targets (25.2%) at a K:T ratio of 5: 1. Finally, Table 1 shows the results of an experiment designed to look for PBl recognising CTL in a polyclonal population of CTL from primed CBA (H2K) mice spleens stimulated with influenza virus in vitro for 5 days. It can be seen that there was very minimal recognition of both PBl and NSl proteins. It appears that PBl -specific CTL are present at a low frequency in this polyclonal CTL population and greater levels of target cell lysis are achieved using a cloned CTL. This report that the polymerase protein PBl can be recognised by cytotbxic T cells confirms that nonglycoprotein nontransmembrane protein components coded for by the viral genome can stimulate these T cells. It is now known that nucleoprotein (a), polymerase protein PB2 (IO), PA and nonstructural protein NSl (7 7), matrix protein (12), and as reported here PBl can be effective target antigens to CTL. Although matrix protein (13), nucleoprotein (14), nonstructural protein (15), and M2 protein (16) have all been unexpectedly detected on the cell surface, the manner in which they are presented to the CTLs in unclear. Transcription and replication of the influenza virus involves a complex set of enzymatic reactions catalysed by the three virion-associated polymerase molecules encoded by the three largest of the eight RNA segments of the influenza virus (17). PBl has been shown to catalyse the addition of free ribonucleotide triphos-
release
CTL- medium
medium
Spontaneous
release)
“Cr
release
In the absence
of
x 1 oo,
release)
phates to the 3’ end of growing transcripts (18). The function of PBl thus places it firmly in the nucleus, but it or a derivative must reach the cell surface in order for CTL recognition to occur. There are two possible explanations for the presence of PBl on the cell surface. First, as the polymerases and nucleoproteins are found in association with the virion RNA (19) this complex must reach the cell surface during budding of virus during productive infection. As NP has been seen on the surface of virus infected cells (14) it is possible that PBl crosses the membrane by the same route. Alternatively, it is possible that PBl is processed by the cell resulting in the presentation of short peptides at the cell surface.
ACKNOWLEDGMENTS This work was supported by the Medical Research Council. We are grateful to Dr. B. Moss and Dr. G. Smith for the gift of recombinant vaccinia viruses.
REFERENCES 1. YAP, K. C., ADA, G. C., and MCKENZIE, I. F. C., Nature (London) 273,238-239 (1978). 2. LIN, Y. L., and ASKONAS, B. A., J. Exp. Med. 154,225-234 (1981). 3. ZWEERINK, H. J., COURTNEIDGE, S. A., SKEHEL, J. J., CRUMPTON, M. I., and ASKONAS, B. A., Nature (London) 267, 354-356 (1977). 4. TOWNSEND, A. R. M., MCMICHAEL, A. J., CARTER, N. P., HUDDLESTON, J. A., and BROWNLEE, G. G., Cell 39, 13-25 (1984).
280
SHORT
COMMUNICATIONS
5. BRACIALE, T. J., BRACIALE, V. L., HENKEL, T. J., SAMBROOK, J.. and GETHING, M. J., J. Exp. Med. 159, 341-354 (1984). 6. YEWDELL, J. W., BENNINK, 1. R., SMITH, G. L.. and Moss, B., Proc. Natl. Acad. Sci. USA 82, 1785-l 789 (1985). 7. BENNINK, J. R., YEWDELL, J. W., SMITH, G. L., MOLLER, C., and Moss, B., Nature (London) 311, 578-579 (1984). 8. TOWNSEND, A. R. M., ROTHBARD, J., GOTCH, F. M., BAHDUR, G., WRAITH, D., and MCMICHAEL, A. J., Cell 44, 959-968 (1986). 9. TOWNSEND, 655-657
A. R. M., (1982).
and SKEHEL, J. J., Nature
(London)
10. BENNINK, J. R.. YEWDELL, J. W., and GERHARD. W., Nature 296, 75-76 (1982).
300,
(London)
11. YEWDELL, J. W., BENNINK, J. R.. GERHARD, W., SMITH, G., and Moss, B., 1. Viral. 61, 1098-l 102 (1987).
12. GOTCH, F. M., MCMICHAEL, A. J.. SMITH G., and Moss, B.. J. Exp. Med. 165,408-416 (1987). 13. BRACIALE, T. J., /. fxp. Med. 146, 673-689 (1977). 14. VIRELIZIER, 1. L., ALLISON, A., OXFORD, J., and SCHILD, G. C., Nature (London) 266, 52-53 (1977). 15. SHAW, M. W., LAMON, E. W., and COMPANS, R. W., Infect. lmmun. 74, 1065-1067 (1981). 76. LAMB, R. A., ZEBEDEE, S. L., and RICHARDSON, C. D., Ce//40,627633 (1985). 17. LAMB, R. A.. and CHOPPIN, P. W., Annu. Rev. Biochem. 52,467506 (1983). 18. BRA~M, J., ULMANENM, I., and KRUG, R. M.. Ce// 34, 609-618 (1983). 19. BISHOP, D. H., ROY, P., BEAN, W. J., and SIMPSON, R. W., J. Viral. 10,689-697 (1972).
160,278-280
(1987)
Specific Recognition
of Influenza
JUDY M. BASTIN,’ Nuffield
Department
of Clinical
Virus Polymerase
Protein
ALAIN R. M. TOWNSEND,
Medicine, Received
John Radcliffe February
Hospital!
13, 1987;
(PBl)
by a Murine
Cytotoxic
AND ANDREW J. MCMICHAEL Headington,
accepted
April
Oxford
OX3 9DU,
Copyright All rights
$3.00
0 1987 by Academic Press. Inc. of reproduction in any form reserved.
Kingdom
influenza virus. virus expressing
by repeated antigenic stimulation in the same conditioned medium. Lytic clones were tested in a standard 6-hr 5’Cr release assay using L cells infected with recombinant vaccinia virus containing influenza cDNA sequences from A/PW8/34 virus (at a multiplicity of infection of 10: 1) and with whole influenza virus. The recombinant vaccinia viruses used were provided by Dr. B. Moss (NIH) and Dr. G. Smith (Cambridge) and contained cDNA corresponding to influenza A/PR/8/34 virus RNA segment 1 (PB2-VAC), segment 2 (PBl -VAC), segment 3 (PA-VAC), segment 4 (H 1-VAC), segment 5 (NP-VAC), segment 7 (M-VAC), and segment 8 (NSlVAC). In addition H2-VAC containing cDNA of segment 4 of AIJAP/305/1957 influenza virus was used. Clone 2-l 6-4 was shown to lyse L cells infected with either influenza A virus A/PR/8/34 (Hl N 1) or A/NT/601 68 (H3N2). However, it did not lyse target cells transfected with NP cDNA of influenza A/NT/60/68 virus. This implied that the cross-reactive clone 2-l 6-4 may recognise some component of influenza virus other than nucleoprotein. It was therefore tested on L cells infected with a series of recombinant vaccinia viruses which each express one of the remaining influenza proteins. Table 1 shows the recognition by clone 2-l 64 (99.7% Ly2 positive by flow cytometric analysis) of L-cell targets infected with PBl-VAC and A/NT/60/68 virus but not of cells infected with vaccinia without influenza cDNA inserts. In addition it shows that this clone did not recognise targets infected with recombinant vaccinia expressing the following influenza proteins: PB2, PA, NP, H2, Hl, NSl, and matrix. Table 1 also shows the recognition by a lo-week-old CBA influenza-specific CTL line 3-9-4, of both PBl and NP inserts but not the others tested. In order to show that the PBl vaccinia-infected targets did not become susceptible to nonspecific killer activity that might be in the T-cell cultures, a C57 Db restricted CTL clone, F5, specific for 1968 NP (4) was used as a negative control. This population of cells did not lyse either PBl
’ To whom requests for reprints should be addressed. * Abbreviations used: CTL, cytotoxic T lymphocytes; PBl , polymerase protein 1 (basic); PB2, polymerase protein 2 (basic); PA, polymerase protein (acidic); Hl, haemagglutinin type 1; H2, haemagglutinin type 2; M, matrix protein; NP, nucleoprotein; NSl, nonstructural protein; VAC vaccinia. 0042-6822187
United
3, 1987
Cytotoxic T-cell clones were raised in CBA mice that recognised both A/X31 and A/JAP/305/1957 Here, we describe one CTL clone that recognises target cells infected with a recombinant vaccinia influenza PBl . a3 1997 Academic press, hc.
Major histocompatibility complex restricted virus specific cytotoxic T lymphocytes (CTL)’ are a prominent feature of host immune responses to many viruses, where they appear to play a role limiting viral replication and dissemination (1, 2). The majority of murine CTL recognises a cross-reactive determinant shared by influenza A but not B viruses, minor populations can distinguish between influenza A subtypes (3). The viral antigens recognised by cross-reactive anti-influenza virus CTL remained largely undefined until recently when it became possible to insert defined viral genes into target cells by DNA transfection (4, 5) and also by the use of recombinant vaccinia virus (6, 7). Use of these methods and more recently use of synthesised peptides of viral proteins has shown that a major population of cross-reactive CTL recognises influenza A virus nucleoprotein (4, 6, 8). Here we describe a murine CTL clone that can recognise target cells that express another internal protein of influenza virus, i.e., polymerase protein PBl . All the methods used to derive and test influenza Aspecific murine CTL have been described in detail elsewhere (8). Briefly, polyclonal cytotoxic lines were established by repeated stimulation in vitro of spleen cells from intraperitoneally primed (A/JAP/305/1957 influenza virus) donor CBA (H2K) mice, with irradiated influenza A/X3 1 or A/PRf8/34 virus-infected syngeneic spleen cells. After two stimulations, T-cell growth factors in the form of supernatant from concanavalin Apulsed rat spleen cells were added to a final concentration of 20% (v/v). In order to raise NP specific clones, CTL were cloned by limiting dilution onto an influenza A/X31 -infected spleen cell feeder layer and maintained
T-Cell Clone
278
SHORT
COMMUNICATIONS TABLE
CV~OTOXICITY Effector KIT ratio
279
1
DATA ON CLONE 2/l 6/4 AND LINE 3/9/4
(PERCENTAGE SPECIFIC “Cr
RELEASE)~
A/NT60/68
V
PBl
PB2
PA
NP
H2
Hl
NSl
Matrix
Uninfected cells
2/l 614 CTL clone
10 5 2.5
29.5 23.1 15.9
2.8 0.0 0.0
39.4 26.8 23.2
4.1 3.2 0.0
1.6 1.7 1.3
6.9 8.0 3.6
0.6 0.1 0.0
8.4 3.3 7.5
4.4 0.8 2.3
5.3 4.0 4.0
NT NT NT
3/g/4 CTL line
10 5 2.5
50.9 41.7 16.0
3.9 0.0 0.0
43.5 23.5 10.1
9.2 4.1 0.0
6.3 4.6 5.2
97.6 50.3 26.3
0.0 0.0 0.0
7.4 5.2 4.5
2.7 2.4 0.9
8.8 0.0 0.3
NT NT NT
5.0 4.5 5.0
8.3 5.6 3.5
2.7 2.4 0.8
3.4 2.7 2.4
19.6 12.3 7.4
NT NT NT
11.3 6.4 3.4
8.1 5.8 4.5
NT NT NT
4.5 3.2 2.3
in medium
alone.
A/PFU8/34 6-Day CBA polyclonal line
15 7.5 3.75
36.4 31.1 18.4
Nore. Each point was measured in duplicate against CTL ranged between 9 and 29% of the Triton release. B Percentage
specific
5’Cr release
was calculated
as
quadriplicate (release
controls
in presenceof (Briton
vaccinia-infected or nonrecombinant vaccinia-infected (0.5% specific release) targets, but did lyse ANT60/68 infected targets (25.2%) at a K:T ratio of 5: 1. Finally, Table 1 shows the results of an experiment designed to look for PBl recognising CTL in a polyclonal population of CTL from primed CBA (H2K) mice spleens stimulated with influenza virus in vitro for 5 days. It can be seen that there was very minimal recognition of both PBl and NSl proteins. It appears that PBl -specific CTL are present at a low frequency in this polyclonal CTL population and greater levels of target cell lysis are achieved using a cloned CTL. This report that the polymerase protein PBl can be recognised by cytotbxic T cells confirms that nonglycoprotein nontransmembrane protein components coded for by the viral genome can stimulate these T cells. It is now known that nucleoprotein (a), polymerase protein PB2 (IO), PA and nonstructural protein NSl (7 7), matrix protein (12), and as reported here PBl can be effective target antigens to CTL. Although matrix protein (13), nucleoprotein (14), nonstructural protein (15), and M2 protein (16) have all been unexpectedly detected on the cell surface, the manner in which they are presented to the CTLs in unclear. Transcription and replication of the influenza virus involves a complex set of enzymatic reactions catalysed by the three virion-associated polymerase molecules encoded by the three largest of the eight RNA segments of the influenza virus (17). PBl has been shown to catalyse the addition of free ribonucleotide triphos-
release
CTL- medium
medium
Spontaneous
release)
“Cr
release
In the absence
of
x 1 oo,
release)
phates to the 3’ end of growing transcripts (18). The function of PBl thus places it firmly in the nucleus, but it or a derivative must reach the cell surface in order for CTL recognition to occur. There are two possible explanations for the presence of PBl on the cell surface. First, as the polymerases and nucleoproteins are found in association with the virion RNA (19) this complex must reach the cell surface during budding of virus during productive infection. As NP has been seen on the surface of virus infected cells (14) it is possible that PBl crosses the membrane by the same route. Alternatively, it is possible that PBl is processed by the cell resulting in the presentation of short peptides at the cell surface.
ACKNOWLEDGMENTS This work was supported by the Medical Research Council. We are grateful to Dr. B. Moss and Dr. G. Smith for the gift of recombinant vaccinia viruses.
REFERENCES 1. YAP, K. C., ADA, G. C., and MCKENZIE, I. F. C., Nature (London) 273,238-239 (1978). 2. LIN, Y. L., and ASKONAS, B. A., J. Exp. Med. 154,225-234 (1981). 3. ZWEERINK, H. J., COURTNEIDGE, S. A., SKEHEL, J. J., CRUMPTON, M. I., and ASKONAS, B. A., Nature (London) 267, 354-356 (1977). 4. TOWNSEND, A. R. M., MCMICHAEL, A. J., CARTER, N. P., HUDDLESTON, J. A., and BROWNLEE, G. G., Cell 39, 13-25 (1984).
280
SHORT
COMMUNICATIONS
5. BRACIALE, T. J., BRACIALE, V. L., HENKEL, T. J., SAMBROOK, J.. and GETHING, M. J., J. Exp. Med. 159, 341-354 (1984). 6. YEWDELL, J. W., BENNINK, 1. R., SMITH, G. L.. and Moss, B., Proc. Natl. Acad. Sci. USA 82, 1785-l 789 (1985). 7. BENNINK, J. R., YEWDELL, J. W., SMITH, G. L., MOLLER, C., and Moss, B., Nature (London) 311, 578-579 (1984). 8. TOWNSEND, A. R. M., ROTHBARD, J., GOTCH, F. M., BAHDUR, G., WRAITH, D., and MCMICHAEL, A. J., Cell 44, 959-968 (1986). 9. TOWNSEND, 655-657
A. R. M., (1982).
and SKEHEL, J. J., Nature
(London)
10. BENNINK, J. R.. YEWDELL, J. W., and GERHARD. W., Nature 296, 75-76 (1982).
300,
(London)
11. YEWDELL, J. W., BENNINK, J. R.. GERHARD, W., SMITH, G., and Moss, B., 1. Viral. 61, 1098-l 102 (1987).
12. GOTCH, F. M., MCMICHAEL, A. J.. SMITH G., and Moss, B.. J. Exp. Med. 165,408-416 (1987). 13. BRACIALE, T. J., /. fxp. Med. 146, 673-689 (1977). 14. VIRELIZIER, 1. L., ALLISON, A., OXFORD, J., and SCHILD, G. C., Nature (London) 266, 52-53 (1977). 15. SHAW, M. W., LAMON, E. W., and COMPANS, R. W., Infect. lmmun. 74, 1065-1067 (1981). 76. LAMB, R. A., ZEBEDEE, S. L., and RICHARDSON, C. D., Ce//40,627633 (1985). 17. LAMB, R. A.. and CHOPPIN, P. W., Annu. Rev. Biochem. 52,467506 (1983). 18. BRA~M, J., ULMANENM, I., and KRUG, R. M.. Ce// 34, 609-618 (1983). 19. BISHOP, D. H., ROY, P., BEAN, W. J., and SIMPSON, R. W., J. Viral. 10,689-697 (1972).