Modulation of specific surface receptors and activation sensitization in primary resting CD4+ T lymphocytes by the Nef protein of HIV-1

Keppler OT, Tibroni N, Venzke S, Rauch S and Fackler OT
Source: J Leukoc Biol
Publication Date: (2006)
Issue: 79(3): 616-27
Research Area:
Immunotherapy / Hematology
Cells used in publication:
T cell, human peripheral blood unstim.
Species: human
Tissue Origin: blood
T cell, human stim.
Species: human
Tissue Origin: blood
Nucleofector® I/II/2b
freshly isolated PBMC were transiently transfected using the nucleofection technology (human T cell Nucleofector kit, 107 cells, 5 µg plasmid DNA, Nucleofector Program U-14, Amaxa Biosystems, Germany), according to the manufacturer's instructions. Immediately after nucleofection, cells were transferred into prewarmed culture medium [500 µl RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, and 1% L-glutamine (all from Gibco, Grand Island, NY)]. Four hours post nucleofection, cells were replated in tissue culture plates, which were uncoated or coated with affinitypurified goat anti-mouse immunoglobulin G (HµL; 10 µg/ml, Jackson ImmunoResearch, West Grove, PA), followed by anti-human CD3 monoclonal antibodies (mAb; 1 µg/ml, clone HIT3a, NA/LE, BD PharMingen, San Diego, CA) and anti-human CD28 mAb (1 µg/ml, clone CD28.2., NA/LE, BD PharMingen). Where indicated, T cell activation was performed by treatment with phorbol 12-myristate 13-acetate (PMA; 50 ng/ml) and ionomycin (1 µM; both from Sigma Chemical Co., St. Louis, MO) or by treatment with phytohemagglutinin- P (PHA-P; 1 µg/ml, Sigma Chemical Co.) and human recombinant IL-2 (20 nM, kind gift from Chiron Corp., Emeryville, CA).
The human immunodeficiency virus type 1 (HIV-1) pathogenicity factor Nef increases viral replication in vivo. In immortalized cell lines, Nef affects the cell surface levels of multiple receptors and signal transduction pathways. Resting CD4(+) T lymphocytes are important targets for HIV-1 infection in vivo--they actively transcribe and express HIV-1 genes and contribute to the local viral burden and long-lived viral reservoirs in patients undergoing antiretroviral therapy. In vitro, this primary cell type has, however, thus far been highly refractory to experimental manipulation, and the biological activities exerted by HIV-1 Nef in these cells are largely unknown. Using nucleofection for gene delivery, we find that Nef induces a drastic and moderate down-regulation of CD4 and major histocompatibility complex type 1 (MHC-I), respectively, but does not alter surface levels of other receptors, the down-modulation of which has been reported in cell line studies. In contrast, Nef markedly up-regulated cell surface levels of the MHC-II invariant chain CD74. The effect of Nef on these three surface receptors was also detected upon HIV-1 infection of activated primary CD4(+) T lymphocytes. Nef expression alone was insufficient to activate resting CD4(+) T lymphocytes, but Nef modestly enhanced the responsiveness of cells to exogenous T cell activation. Consistent with such a signal transduction activity, a subpopulation of Nef localized to lipid raft clusters at the plasma membrane. This study establishes the analysis of Nef functions in these primary HIV target cells. Our data support the involvement of modulation of a defined set of cell surface receptors and sensitization to activation rather than an autonomous activation function in the role of Nef in HIV-1 pathogenesis.