ABSTRACT
Therapeutic vaccination aims at a strong stimulation of antigen-specific CD8(+) T-cells, so that they differentiate into effectors active in vivo against antigenic targets. Two adenovirus vectors (Ad) encoding two HLA-A*0201-restricted HIV epitope sequences (pol 476 and pol 589) were constructed. The Ad differ by the presence or absence of a ubiquitin monomer sequence (AdUb(+) and AdUb(-)). The effect of transgene product ubiquitination was analyzed on (1) in vivo, the immunization of Ad vaccinated HLA-A*0201 humanized HHD mice and (2) in vitro, the presentation of the transgene encoded peptides by transduced human dendritic cells (DC). In vivo, we found that immunization of humanized HHD mice with AdUb(+) elicited a transgene product-specific interferon (INF)-gamma CD8(+) T-cell response detectable by enzyme-linked immunospot (ELISPOT), whereas the AdUb(-) construction did not. Antigen-specific cytotoxic T lymphocytes (CTL) were also generated in HHD mice immunized with AdUb(+) and not with AdUb(-). In vitro, using human AdUb(+)-transduced DC, a sizeable expansion of pol 476 and pol 589 tetramer positive CD8(+) T cells as well as CD8(+) CTL were obtained in healthy donors. Compared to AdUb(-)-transduced DC, AdUb(+)-transduced DC triggered a higher number of pol 476-specific IFN-gamma-secreting CD8(+) T cells. In agreement, AdUb(+) transduced DC, used as target in a (51)Cr-release assay, were more efficiently lysed by peptide-specific CTL than AdUb(-)-transduced DC. In conclusion, the addition of an ubiquitin sequence to the adenoviral transgene, used as an antigen source, resulted in both in vivo enhanced CD8(+) T-cell immunogenicity in HHD mice and in vitro increased HLA class I-restricted presentation of encoded peptides by human DC.
Subject(s)
Adenoviruses, Human/genetics , Dendritic Cells/immunology , Gene Transfer Techniques , Histocompatibility Antigens Class I/immunology , Immunotherapy, Active/methods , Ubiquitin/metabolism , Adenoviruses, Human/immunology , Animals , Antigen-Presenting Cells/immunology , CD8-Positive T-Lymphocytes/immunology , Cell Line, Tumor , Epitopes , Genetic Therapy , Humans , Interferon-gamma/immunology , K562 Cells , Mice , Mice, Knockout , Mice, Transgenic , Transduction, Genetic , TransgenesABSTRACT
We observed in the same patient the development of a tonsil mucosa-associated lymphoid tissue-type lymphoma in 1994, a mediastinal Hodgkin's disease in 1998, and a colonic CD30+ anaplastic diffuse large B-cell lymphoma in 2000. A same-sized FR3-JH fragment was demonstrated by polymerase chain reaction, both at the level of total DNA and of single micromanipulated cells, showing monocytoid, Reed-Sternberg, or anaplastic morphology. Moreover, an identical IgH nucleotide sequence was detected in mucosa-associated lymphoid tissue-type lymphoma and colonic CD30+ anaplastic diffuse large B-cell lymphoma, whereas mediastinal Hodgkin's disease IgH rearrangement involved different VH and JH genes. CD30+ Reed-Sternberg and diffuse large B-cell lymphoma cells contained Epstein-Barr virus EBER sequences that were not observed at the level of mucosa-associated lymphoid tissue-type lymphoma. Therefore, Epstein-Barr virus infection may have played a role in diffuse large B-cell lymphoma transformation of mucosa-associated lymphoid tissue-type lymphoma and in the lymphomagenesis of Hodgkin's disease. In addition to their different clonal origin, Reed-Sternberg cells of Hodgkin's disease expressed a CD15+, CD20+ (rare cells), CD30+, Oct-2-, EBNA2-, LMP1+ phenotype, whereas anaplastic and Reed-Sternberg-like cells of diffuse large B-cell lymphoma were CD15-, CD20+, CD30+, Oct-2+, EBNA2+, and LMP1+. Interestingly, we also detected scattered CD30+ Epstein-Barr virus- large cells with prominent nucleoli in the initial tonsil mucosa-associated lymphoid tissue-type lymphoma, suggesting that these cells could be prone to Epstein-Barr virus infection and/or large cell transformation.
