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. 1998 Feb 2;187(3):445-50.
doi: 10.1084/jem.187.3.445.

Murine cytotoxic T lymphocytes recognize an epitope in an EBNA-1 fragment, but fail to lyse EBNA-1-expressing mouse cells

S Mukherjee  1 , P Trivedi, D M Dorfman, G Klein, A Townsend
Affiliations

Murine cytotoxic T lymphocytes recognize an epitope in an EBNA-1 fragment, but fail to lyse EBNA-1-expressing mouse cells

S Mukherjee et al. J Exp Med. .

Abstract

Major histocompatibility complex class I-restricted cytotoxic T lymphocytes (CTLs) specific for epitopes within eight of the nine Epstein Barr Virus (EBV)-encoded latency-associated proteins have been recovered from EBV-infected human subjects by restimulation of lymphocytes in vitro. However, human class I-restricted CTL responses capable of recognizing EBNA-1 expressing cells were not detected in these studies. We have raised a murine CTL line that recognizes an epitope within EBNA-1 by immunizing mice with a vaccinia virus encoding a COOH-terminal EBNA-1 fragment. This novel CTL line was used to investigate whether the epitope (positions 509-517 in EBNA-1, presented through Kd) was presented to CTL by mouse cells expressing full-length EBNA-1 or a deletion mutant of EBNA-1, lacking the Glycine-Alanine (Gly-Ala)-rich region. Cells expressing full-length EBNA-1 are not lysed by the CTL line, whereas cells expressing the Gly-Ala deletion mutant are recognized. These results suggest that epitopes from full-length EBNA-1 are poorly presented, and that the Gly-Ala-rich region is responsible for this phenomenon. The inefficient presentation of EBNA-1-derived epitopes may explain the absence or rarity of EBNA-1-specific CTLs in vivo, a strategy that may allow EBV to maintain persistence within the immunocompetent host without being eliminated by CTLs.

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Figures

Figure 2
Figure 2
(a) Constructs used to transfect P815 cells. (Top) Full-length EBNA-1 of the B-95-8 sequence (641 amino acids total). Hatched region (93–328) shows the Gly-Ala–rich region. NLS, nuclear localisation sequence as described in reference . The position of the V9L epitope is 509–517 (indicated as V9L). (Bottom) ΔEBNA-1: residues 93–325 have been deleted, but the rest of EBNA-1 is unaffected. (b, i) Western Blot analysis of transfected cells using the Rbt-EBNA-1 antibody (11). Cells are as labeled. A 72-kD band is detected in P815/EBNA-1 cells; based on reactivity with the antiserum and the size of EBNA-1 detected in the 721 LCL line (lane 4), this is EBNA-1. A 43-kD band is detected in P815 cells transfected with EBNA-1 lacking the Gly-Ala repeat. (b, ii) Western Blot using the P-107 serum (12) that reacts against the Gly-Ala repeat only. Lanes 2 and 3 indicate P815 cells transfected with vectors alone (i.e., no EBNA-1). Lane 6, NAD20 cells are EBV-transformed LCLs. (c) Cell staining with Rbt-EBNA-1 serum (11) reactive against EBNA-1. Nuclear staining is indicated with N and arrow. Cells are as indicated in legend and text.
Figure 2
Figure 2
(a) Constructs used to transfect P815 cells. (Top) Full-length EBNA-1 of the B-95-8 sequence (641 amino acids total). Hatched region (93–328) shows the Gly-Ala–rich region. NLS, nuclear localisation sequence as described in reference . The position of the V9L epitope is 509–517 (indicated as V9L). (Bottom) ΔEBNA-1: residues 93–325 have been deleted, but the rest of EBNA-1 is unaffected. (b, i) Western Blot analysis of transfected cells using the Rbt-EBNA-1 antibody (11). Cells are as labeled. A 72-kD band is detected in P815/EBNA-1 cells; based on reactivity with the antiserum and the size of EBNA-1 detected in the 721 LCL line (lane 4), this is EBNA-1. A 43-kD band is detected in P815 cells transfected with EBNA-1 lacking the Gly-Ala repeat. (b, ii) Western Blot using the P-107 serum (12) that reacts against the Gly-Ala repeat only. Lanes 2 and 3 indicate P815 cells transfected with vectors alone (i.e., no EBNA-1). Lane 6, NAD20 cells are EBV-transformed LCLs. (c) Cell staining with Rbt-EBNA-1 serum (11) reactive against EBNA-1. Nuclear staining is indicated with N and arrow. Cells are as indicated in legend and text.
Figure 2
Figure 2
(a) Constructs used to transfect P815 cells. (Top) Full-length EBNA-1 of the B-95-8 sequence (641 amino acids total). Hatched region (93–328) shows the Gly-Ala–rich region. NLS, nuclear localisation sequence as described in reference . The position of the V9L epitope is 509–517 (indicated as V9L). (Bottom) ΔEBNA-1: residues 93–325 have been deleted, but the rest of EBNA-1 is unaffected. (b, i) Western Blot analysis of transfected cells using the Rbt-EBNA-1 antibody (11). Cells are as labeled. A 72-kD band is detected in P815/EBNA-1 cells; based on reactivity with the antiserum and the size of EBNA-1 detected in the 721 LCL line (lane 4), this is EBNA-1. A 43-kD band is detected in P815 cells transfected with EBNA-1 lacking the Gly-Ala repeat. (b, ii) Western Blot using the P-107 serum (12) that reacts against the Gly-Ala repeat only. Lanes 2 and 3 indicate P815 cells transfected with vectors alone (i.e., no EBNA-1). Lane 6, NAD20 cells are EBV-transformed LCLs. (c) Cell staining with Rbt-EBNA-1 serum (11) reactive against EBNA-1. Nuclear staining is indicated with N and arrow. Cells are as indicated in legend and text.
Figure 2
Figure 2
(a) Constructs used to transfect P815 cells. (Top) Full-length EBNA-1 of the B-95-8 sequence (641 amino acids total). Hatched region (93–328) shows the Gly-Ala–rich region. NLS, nuclear localisation sequence as described in reference . The position of the V9L epitope is 509–517 (indicated as V9L). (Bottom) ΔEBNA-1: residues 93–325 have been deleted, but the rest of EBNA-1 is unaffected. (b, i) Western Blot analysis of transfected cells using the Rbt-EBNA-1 antibody (11). Cells are as labeled. A 72-kD band is detected in P815/EBNA-1 cells; based on reactivity with the antiserum and the size of EBNA-1 detected in the 721 LCL line (lane 4), this is EBNA-1. A 43-kD band is detected in P815 cells transfected with EBNA-1 lacking the Gly-Ala repeat. (b, ii) Western Blot using the P-107 serum (12) that reacts against the Gly-Ala repeat only. Lanes 2 and 3 indicate P815 cells transfected with vectors alone (i.e., no EBNA-1). Lane 6, NAD20 cells are EBV-transformed LCLs. (c) Cell staining with Rbt-EBNA-1 serum (11) reactive against EBNA-1. Nuclear staining is indicated with N and arrow. Cells are as indicated in legend and text.
Figure 1
Figure 1
(a) Scheme for rescue of peptide-specific CTLs by coculturing immunized splenocytes with naive splenocytes pulsed with candidate peptides (see text). (1) Identification of candidate epitopes. (2) Construction of vaccinia virus expressing the EBNA-1 (505–583) fragment (called VVΔEB-1) containing the candidate epitopes. (3) Immunization of mice and recovery of CTLs. (b) Mouse strains initially immunized with VVΔEB-1 and the peptides used to restimulate them in vitro. (Column 1) Candidate epitopes derived from the COOH-terminal of EBNA-1 using consensus motifs described in reference . (Column 2) Class I molecule for which the candidate peptide in column 1 carries the consensus motif (and strains immunized with VVΔEB-1). For example, VYGGSKTSL contains a Kd motif (i.e., Y at position 2, and L at position 9, underlined). Splenocytes from BALB/c mice (H2-d) immunized with VVΔEB-1 were restimulated with splenocytes pulsed with the V9L peptide, as described in a. Cocultures restimulated on peptides (as in column 1) were used as effectors in a Cr–release assay using peptide-pulsed cells as targets (see below for the target cells). (Column 3) Specific lysis above background in 51Cr release assays 5 d after in vitro restimulation with peptide-pulsed cells, was obtained for each of the effector cultures. Target cells used: P815 for all H2-d, L929 for H2-k, and EL-4 for H2-b. (c) V9L CTLs are peptide specific and MHC restricted. P815 cells were pulsed with the peptide dose indicated (in nM) and used as targets in Cr–release assay. K/T ratio was 20:1. Unpulsed cells (0 nM) are not lysed. Hatched bar shows L/Db cells that were pulsed with V9L peptide; these cells do not express Kd and were not lysed. The x-axis represents P815 cells pulsed with peptide at concentration indicated (in nM) and used as target for V9L CTLs.
Figure 1
Figure 1
(a) Scheme for rescue of peptide-specific CTLs by coculturing immunized splenocytes with naive splenocytes pulsed with candidate peptides (see text). (1) Identification of candidate epitopes. (2) Construction of vaccinia virus expressing the EBNA-1 (505–583) fragment (called VVΔEB-1) containing the candidate epitopes. (3) Immunization of mice and recovery of CTLs. (b) Mouse strains initially immunized with VVΔEB-1 and the peptides used to restimulate them in vitro. (Column 1) Candidate epitopes derived from the COOH-terminal of EBNA-1 using consensus motifs described in reference . (Column 2) Class I molecule for which the candidate peptide in column 1 carries the consensus motif (and strains immunized with VVΔEB-1). For example, VYGGSKTSL contains a Kd motif (i.e., Y at position 2, and L at position 9, underlined). Splenocytes from BALB/c mice (H2-d) immunized with VVΔEB-1 were restimulated with splenocytes pulsed with the V9L peptide, as described in a. Cocultures restimulated on peptides (as in column 1) were used as effectors in a Cr–release assay using peptide-pulsed cells as targets (see below for the target cells). (Column 3) Specific lysis above background in 51Cr release assays 5 d after in vitro restimulation with peptide-pulsed cells, was obtained for each of the effector cultures. Target cells used: P815 for all H2-d, L929 for H2-k, and EL-4 for H2-b. (c) V9L CTLs are peptide specific and MHC restricted. P815 cells were pulsed with the peptide dose indicated (in nM) and used as targets in Cr–release assay. K/T ratio was 20:1. Unpulsed cells (0 nM) are not lysed. Hatched bar shows L/Db cells that were pulsed with V9L peptide; these cells do not express Kd and were not lysed. The x-axis represents P815 cells pulsed with peptide at concentration indicated (in nM) and used as target for V9L CTLs.
Figure 1
Figure 1
(a) Scheme for rescue of peptide-specific CTLs by coculturing immunized splenocytes with naive splenocytes pulsed with candidate peptides (see text). (1) Identification of candidate epitopes. (2) Construction of vaccinia virus expressing the EBNA-1 (505–583) fragment (called VVΔEB-1) containing the candidate epitopes. (3) Immunization of mice and recovery of CTLs. (b) Mouse strains initially immunized with VVΔEB-1 and the peptides used to restimulate them in vitro. (Column 1) Candidate epitopes derived from the COOH-terminal of EBNA-1 using consensus motifs described in reference . (Column 2) Class I molecule for which the candidate peptide in column 1 carries the consensus motif (and strains immunized with VVΔEB-1). For example, VYGGSKTSL contains a Kd motif (i.e., Y at position 2, and L at position 9, underlined). Splenocytes from BALB/c mice (H2-d) immunized with VVΔEB-1 were restimulated with splenocytes pulsed with the V9L peptide, as described in a. Cocultures restimulated on peptides (as in column 1) were used as effectors in a Cr–release assay using peptide-pulsed cells as targets (see below for the target cells). (Column 3) Specific lysis above background in 51Cr release assays 5 d after in vitro restimulation with peptide-pulsed cells, was obtained for each of the effector cultures. Target cells used: P815 for all H2-d, L929 for H2-k, and EL-4 for H2-b. (c) V9L CTLs are peptide specific and MHC restricted. P815 cells were pulsed with the peptide dose indicated (in nM) and used as targets in Cr–release assay. K/T ratio was 20:1. Unpulsed cells (0 nM) are not lysed. Hatched bar shows L/Db cells that were pulsed with V9L peptide; these cells do not express Kd and were not lysed. The x-axis represents P815 cells pulsed with peptide at concentration indicated (in nM) and used as target for V9L CTLs.
Figure 3
Figure 3
V9L CTLs used as effectors in 51Cr–release assays. Targets: P815/EBNA-1 (open circles), P815/ΔEBNA-1 (filled squares), P815 with no peptide (open triangles), and a P815 + V9L peptide pulsed at 10 μm (filled circles).
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