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CD8+ T cell responses to lytic EBV infection: late antigen specificities as subdominant components of the total response.

ABSTRACT: EBV elicits primary CD8(+) T cell responses that, by T cell cloning from infectious mononucleosis (IM) patients, appear skewed toward immediate early (IE) and some early (E) lytic cycle proteins, with late (L) proteins rarely targeted. However, L Ag-specific responses have been detected regularly in polyclonal T cell cultures from long-term virus carriers. To resolve this apparent difference between responses to primary and persistent infection, 13 long-term carriers were screened in ex vivo IFN-? ELISPOT assays using peptides spanning the two IE, six representative E, and seven representative L proteins. This revealed memory CD8 responses to 44 new lytic cycle epitopes that straddle all three protein classes but, in terms of both frequency and size, maintain the IE > E > L hierarchy of immunodominance. Having identified the HLA restriction of 10 (including 7 L) new epitopes using memory CD8(+) T cell clones, we looked in HLA-matched IM patients and found such reactivities but typically at low levels, explaining why they had gone undetected in the original IM clonal screens. Wherever tested, all CD8(+) T cell clones against these novel lytic cycle epitopes recognized lytically infected cells naturally expressing their target Ag. Surprisingly, however, clones against the most frequently recognized L Ag, the BNRF1 tegument protein, also recognized latently infected, growth-transformed cells. We infer that BNRF1 is also a latent Ag that could be targeted in T cell therapy of EBV-driven B-lymphoproliferative disease.

SUBMITTER: Abbott RJ

PROVIDER: S-EPMC5580796 | biostudies-literature | 2013 Dec

REPOSITORIES: biostudies-literature

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CD8+ T cell responses to lytic EBV infection: late antigen specificities as subdominant components of the total response.

Abbott Rachel J M RJ Quinn Laura L LL Leese Alison M AM Scholes Harry M HM Pachnio Annette A Rickinson Alan B AB

Journal of immunology (Baltimore, Md. : 1950) 20131021 11

EBV elicits primary CD8(+) T cell responses that, by T cell cloning from infectious mononucleosis (IM) patients, appear skewed toward immediate early (IE) and some early (E) lytic cycle proteins, with late (L) proteins rarely targeted. However, L Ag-specific responses have been detected regularly in polyclonal T cell cultures from long-term virus carriers. To resolve this apparent difference between responses to primary and persistent infection, 13 long-term carriers were screened in ex vivo IFN ...[more]

PMID: 24146041

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Project description:Long term carriers were shown to generate robust polyfunctional T cell (PFC) responses against lytic and latent antigens of Epstein-Barr virus (EBV). However, the time of emergence of PFC responses against EBV antigens, pattern of immunodominance and difference between CD4+ and CD8+ T cell responses during various stages of EBV infection are not clearly understood. A longitudinal study was performed to assess the development of antigen-specific PFC responses in children diagnosed to have primary symptomatic (infectious mononucleosis [IM]) and asymptomatic (AS) EBV infection. Evaluation of IFN-? secreting CD8+ T cell responses upon stimulation by HLA class I-specific peptides of EBV lytic and latent proteins by ELISPOT assay followed by assessment of CD4+ and CD8+ PFC responses upon stimulation by a panel of overlapping EBV peptides for co-expression of IFN-?, TNF-?, IL-2, perforin and CD107a by flow cytometry were performed. Cytotoxicity of T cells against autologous lymphoblastoid cell lines (LCLs) as well as EBV loads in PBMC and plasma were also determined. Both IM and AS patients had elevated PBMC and plasma viral loads which declined steadily during a 12-month period from the time of diagnosis whilst decrease in the magnitude of CD8+ T cell responses toward EBV lytic peptides in contrast to increase toward latent peptides was shown with no significant difference between those of IM and AS patients. Both lytic and latent antigen-specific CD4+ and CD8+ T cells demonstrated polyfunctionality (defined as greater or equal to three functions) concurrent with enhanced cytotoxicity against autologous LCLs and steady decrease in plasma and PBMC viral loads over time. Immunodominant peptides derived from BZLF1, BRLF1, BMLF1 and EBNA3A-C proteins induced the highest proportion of CD8+ as well as CD4+ PFC responses. Diverse functional subtypes of both CD4+ and CD8+ PFCs were shown to emerge at 6-12 months. In conclusion, EBV antigen-specific CD4+ and CD8+ PFC responses emerge during the first year of primary EBV infection, with greatest responses toward immunodominant epitopes in both lytic and latent proteins, correlating to steady decline in PBMC and plasma viral loads.

Project description:IntroductionEpstein-Barr virus (EBV) is a widely spread pathogen associated with lymphoproliferative diseases, B/ T/ NK cell lymphomas, nasopharyngeal carcinoma (NPC) and gastric carcinoma (GC). EBV lytic reactivations contribute to the genomic instability, inflammation and tumorigenesis of NPC, promoting cancer progression. Patients with NPC refractory to standard therapies show dismal survival. EBV gp350 is an envelope protein detectable in NPC specimens intracellularly and on the cell membrane of malignant cells, and is a potential viral antigen for T cell-directed immunotherapies. The potency of T cells engineered with a chimeric antigen receptor (CAR) targeting gp350 against EBV+ lymphoproliferative disease was previously shown.MethodsHere, we advanced towards preclinical and non-clinical developments of this virus-specific CAR-T cell immunotherapy against NPC. Different gp350CAR designs were inserted into a lentiviral vector (LV) backbone.ResultsA construct expressing the scFv 7A1-anti-gp350 incorporating the CD8 transmembrane and CD28.CD3ζ signaling domain (ZT002) was selected. High titer ZT002 (~1x108 TU/ml) was manufactured in HEK 293T/17 suspension cells in serum free media as large-scale production under good manufacturing practices (GMP). A LV multiplicity of infection (MOI) of 1 resulted in high frequencies of functional gp350CAR+ T cells (>70%) at a low (<2) vector copy numbers in the genome. ZT002 was therefore used to establish gp350CAR-T batch run production methods. GMP upscaling and validation of T cell transduction and expansion in several runs resulted in average 3x109 gp350CAR-T cells per batch. >80% CD3+ gp350CAR-T cells bound to purified gp350 protein. In vitro cytotoxicity and cytokine secretion assays (IFN-γ and TNF-α) confirmed the specificity of gp350CAR-T cells against gp350+ NPC, GC and lymphoma cell targets. Immunocompromised B-NDG mice (NOD.CB17-PrkdcscidIl2rgtm1/Bcgen) were challenged s.c. with a EBV+ NPC C666.1 cell line expressing gp350 and then treated with escalating doses of gp350CAR-T cells or with non-transduced T cells. gp350CAR-T cells promoted antitumor responses, bio-distributed in several tissues, infiltrated in tumors and rejected gp350+ tumor cells.DiscussionThese results support the use of gp350CAR-T cells generated with ZT002 as an Innovative New Drug to treat patients with solid and liquid EBV-associated malignancies.

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CD8+ T cell responses to lytic EBV infection: late antigen specificities as subdominant components of the total response.

Publications

CD8+ T cell responses to lytic EBV infection: late antigen specificities as subdominant components of the total response.

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