V beta activation by HIV Nef protein: detection by a simple amplification procedure.
We previously reported that Nef protein from human immunodeficiency virus (HIV) has superantigen properties. However, we were unable to consistently demonstrate V beta-specific expansion by Nef using flow cytometry, possibly due to its lower mitogenic activity compared to prototypic superantigens. Therefore, we developed a simple amplification detection method using immobilized anti-V beta antibodies. Human peripheral blood mononuclear cells from adult donors were treated for 24 h with Nef and restimulated with immobilized anti-V beta antibodies for an additional 72 h. Significant expansion of V beta 5.3 and V beta 18 T-cells were detected in Nef-treated cultures, with expansion of V beta 18 occurring with all donors tested, and V beta 5.3 expansion occurring in 50% of the donors. Variable responses were obtained with V beta 2, V beta 3, and V beta 9. These results were confirmed using the more time-consuming method of reverse transcriptase-polymerase chain reaction (RT-PCR). Thus, this novel, reproducible, and relatively simple method can detect V beta-specific expansion by weak superantigens.
Tanabe
T
T
Department of Microbiology and Cell Science, University of Florida, Gainesville 32611, USA.
Torres
B A
BA
Subramaniam
P S
PS
Johnson
H M
HM
D000328:Adult; D015693:Gene Products, nef / Q000276:immunology / Q000502:physiology*; D006678:HIV / Q000276:immunology / Q000502:physiology*; D006801:Humans; D008213:Lymphocyte Activation / Q000187:drug effects; D016133:Polymerase Chain Reaction / Q000379:methods*; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000378:metabolism*; D015203:Reproducibility of Results; D018089:Superantigens / Q000276:immunology / Q000502:physiology; D016176:T-Lymphocyte Subsets / Q000276:immunology / Q000378:metabolism; D054311:nef Gene Products, Human Immunodeficiency Virus
D015693:Gene Products, nef; D016693:Receptors, Antigen, T-Cell, alpha-beta; D018089:Superantigens; D054311:nef Gene Products, Human Immunodeficiency Virus