CD4+ T cells modulate the magnitude and durability of CTL responses in vivo, and may serve as eff... more CD4+ T cells modulate the magnitude and durability of CTL responses in vivo, and may serve as effector cells in the tumour microenvironment. In order to identify the tumour epitopes recognized by tumour-reactive human CD4+ T cells, we combined the use of an HLA-DR4/peptide binding algorithm with an IFN-gamma ELISPOT assay. Two known and three novel CD4+ T cell epitopes derived from the gp 100/pmel17 and tyrosinase melanocyte-associated antigens were confirmed or identified. Of major interest, we determined that freshly-isolated PBMC frequencies of Th1-type CD4+ T recognizing these peptides are frequently elevated in HLA-DR4+ melanoma patients (but not normal donors) that are currently disease-free as a result of therapeutic intervention. Epitope-specific CD4+ T cells from normal DR4+ donors could be induced, however, after in vitro stimulation with autologous dendritic cell pulsed with antigens (peptides or antigen-positive melanoma lysates) or infected with recombinant vaccinia virus encoding the relevant antigen. Peptide-reactive CD4+ T cells also recognized HLA-DR4+ melanoma cell lines that constitutively express the relevant antigen. Based on these data, these epitopes may serve as potent vaccine components to promote clinically-relevant Th1-type CD4+ T cell effector function in situ.
Objective. Circumstantial evidence links retroviruses (RVs) with human autoimmune diseases. The a... more Objective. Circumstantial evidence links retroviruses (RVs) with human autoimmune diseases. The aim of the present study was to obtain direct evidence of RV gene expression in rheumatoid arthritis (RA).Methods. Synovial samples were obtained from patients with RA, patients with osteoarthritis (OA), and normal control subjects. Reverse transcriptionpolymerase chain reaction (RT-PCR) was performed using synovial RNA and primers to conserved sequences in the polymerase (pol) genes of known RVs.Results. PCR products (n = 857) were cloned and sequenced. Multiple pol transcripts, many with open reading frames, were expressed in every sample. Sequences were aligned and classified into 6 families (F1-F6) that contained 33 groups of known and unknown endogenous RVs (ERVs), each distinguished by a specific, deduced peptide motif. The frequency of sequences in each family was similar between RA, OA, and normal synovial tissue, but differed significantly in RA synovial fluid cells. F1 sequences (undefined, but related to murine and primate type C RVs) were lower in frequency, F2 (ERV-9-related), F4 (HERV-K-related), and F6 (HERV-L-related) sequences were higher in frequency, and F3 (RTVL-H-related) sequences were not detected, in the RA synovial fluid cells compared with the RA synovial tissues.Conclusion. Multiple ERVs are expressed in normal and diseased synovial compartments, but specific transcripts can be differentially expressed in RA.
CD4+ T cells modulate the magnitude and durability of CTL responses in vivo, and may serve as eff... more CD4+ T cells modulate the magnitude and durability of CTL responses in vivo, and may serve as effector cells in the tumour microenvironment. In order to identify the tumour epitopes recognized by tumour-reactive human CD4+ T cells, we combined the use of an HLA-DR4/peptide binding algorithm with an IFN-gamma ELISPOT assay. Two known and three novel CD4+ T cell epitopes derived from the gp 100/pmel17 and tyrosinase melanocyte-associated antigens were confirmed or identified. Of major interest, we determined that freshly-isolated PBMC frequencies of Th1-type CD4+ T recognizing these peptides are frequently elevated in HLA-DR4+ melanoma patients (but not normal donors) that are currently disease-free as a result of therapeutic intervention. Epitope-specific CD4+ T cells from normal DR4+ donors could be induced, however, after in vitro stimulation with autologous dendritic cell pulsed with antigens (peptides or antigen-positive melanoma lysates) or infected with recombinant vaccinia virus encoding the relevant antigen. Peptide-reactive CD4+ T cells also recognized HLA-DR4+ melanoma cell lines that constitutively express the relevant antigen. Based on these data, these epitopes may serve as potent vaccine components to promote clinically-relevant Th1-type CD4+ T cell effector function in situ.
Objective. Circumstantial evidence links retroviruses (RVs) with human autoimmune diseases. The a... more Objective. Circumstantial evidence links retroviruses (RVs) with human autoimmune diseases. The aim of the present study was to obtain direct evidence of RV gene expression in rheumatoid arthritis (RA).Methods. Synovial samples were obtained from patients with RA, patients with osteoarthritis (OA), and normal control subjects. Reverse transcriptionpolymerase chain reaction (RT-PCR) was performed using synovial RNA and primers to conserved sequences in the polymerase (pol) genes of known RVs.Results. PCR products (n = 857) were cloned and sequenced. Multiple pol transcripts, many with open reading frames, were expressed in every sample. Sequences were aligned and classified into 6 families (F1-F6) that contained 33 groups of known and unknown endogenous RVs (ERVs), each distinguished by a specific, deduced peptide motif. The frequency of sequences in each family was similar between RA, OA, and normal synovial tissue, but differed significantly in RA synovial fluid cells. F1 sequences (undefined, but related to murine and primate type C RVs) were lower in frequency, F2 (ERV-9-related), F4 (HERV-K-related), and F6 (HERV-L-related) sequences were higher in frequency, and F3 (RTVL-H-related) sequences were not detected, in the RA synovial fluid cells compared with the RA synovial tissues.Conclusion. Multiple ERVs are expressed in normal and diseased synovial compartments, but specific transcripts can be differentially expressed in RA.
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Papers by Vladimir Brusic