Induction of antigen-specific CTL by recombinant HIV trans-activating fusion protein-pulsed human monocyte-derived dendritic cells.
Several systems have been tested for introduction of Ags into human dendritic cells (DC). Most of them to date, however, are complex and possess limited efficiency. Recent advances in HIV trans-activating (TAT) fusion protein technology permit extremely high transduction efficiencies for a majority of mammalian cell types. Here we report our attempts to develop a simple, but highly efficient, protocol for loading of antigenic protein into DC using TAT fusion technology. A TAT-minigene fusion protein was generated, encoding both the HLA-A2-restricted influenza matrix protein-derived epitope (GILVFTFTL, Flu-M1) and a melanoma Ag gp100-derived modified epitope (YLEPGPVTV, G9(280)-9V). In addition, both a TAT-Her2/neu extracellular domain (ECD) fusion protein and a TAT-green fluorescence protein fusion protein were generated. Over 95% of DC stained positively for TAT-green fluorescence protein within 20 min of coculture. DC treated with TAT-minigene were efficiently recognized by both Flu-M1 and G9(280)-9V-specific T cells in cytotoxicity assays and IFN-gamma ELISPOT assays. In contrast, DC pulsed with minigene fusion protein lacking TAT were either poorly recognized or not recognized by the T cells. DC pulsed with TAT-minigene also efficiently induced Flu-M1-specific T cells from naive lymphocytes. Similarly, DC treated with TAT-Her2/neu ECD stimulated patient-derived lymphocytes that specifically recognized Her2/neu(+) ovarian and breast cancer cell lines. The CTL induced by TAT-Her2/neu ECD-pulsed DC specifically recognized the Her2/neu ECD-derived immunogenic peptide E75 (KIFGSLAFL). Our data suggest that TAT fusion proteins efficiently transduce DC and induce Ag-specific T cells. This could prove to be a useful method for treatment of infectious diseases and cancer.
Tanaka
Yoshiyuki
Y
Department of Surgery, Biologic Cancer Therapy Program, and Alvin J. Siteman Cancer Center, Washington University School of Medicine, St. Louis, MO 63110,USA.
Dowdy
Steven F
SF
Linehan
David C
DC
Eberlein
Timothy J
TJ
Goedegebuure
Peter S
PS
D002460:Cell Line; D003602:Cytotoxicity, Immunologic / Q000235:genetics; D003713:Dendritic Cells / Q000166:cytology / Q000276:immunology* / Q000378:metabolism; D018984:Epitopes, T-Lymphocyte / Q000235:genetics / Q000276:immunology*; D005110:Extracellular Space / Q000235:genetics / Q000502:physiology; D015696:Gene Products, tat / Q000235:genetics* / Q000502:physiology; D015497:HIV-1 / Q000235:genetics* / Q000276:immunology; D015789:HLA-A2 Antigen / Q000276:immunology; D006801:Humans; D020014:K562 Cells; D008213:Lymphocyte Activation / Q000235:genetics; D008562:Membrane Glycoproteins / Q000235:genetics / Q000502:physiology; D009000:Monocytes / Q000166:cytology / Q000276:immunology*; D009363:Neoplasm Proteins / Q000235:genetics / Q000502:physiology; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary / Q000235:genetics; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism / Q000502:physiology*; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*; D014161:Transduction, Genetic; D014407:Tumor Cells, Cultured; D014763:Viral Matrix Proteins / Q000235:genetics / Q000502:physiology; D058951:gp100 Melanoma Antigen; D054322:tat Gene Products, Human Immunodeficiency Virus
D018984:Epitopes, T-Lymphocyte; D015696:Gene Products, tat; D015789:HLA-A2 Antigen; D008562:Membrane Glycoproteins; D009363:Neoplasm Proteins; D018719:Erb-b2 Receptor Tyrosine Kinases; D011993:Recombinant Fusion Proteins; D014763:Viral Matrix Proteins; D058951:gp100 Melanoma Antigen; D054322:tat Gene Products, Human Immunodeficiency Virus; C031540:M-protein, influenza virus; C488936:M1 protein, Influenza A virus; C548637:PMEL protein, human