Interleukin-2 production by polyfunctional HIV-1-specific CD8 T cells is associated with enhanced viral suppression.

BACKGROUND: Assays to measure the induction of HIV-1-specific CD8 T-cell responses often rely on measurements of indirect effector function such as chemokine and cytokine production, which may not reflect direct elimination of an invading pathogen. Assessment of the functional ability of CD8 T cells to suppress HIV-1 replication has been viewed as a surrogate marker of an effectual immune response. To further investigate this, we measured the capacity of virus-specific CD8 T cells to inhibit HIV-1 replication in an in vitro suppression assay. METHODS: We expanded 15 epitope-specific CD8 T-cell lines from peripheral blood mononuclear cells of chronically HIV--infected progressors (n = 5) and controllers (n = 4) who were not on antiretroviral therapy. Cell lines were tested for their ability to produce effector molecules (CD107a, IL-2, IFN-γ, TNF-α, perforin) and suppress virus replication in autologous CD4 T cells. RESULTS: CD8 T-cell lines from both progressors and controllers had largely similar effector function profiles as determined by intracellular cytokine staining. In contrast, we observed that CD8 T-cell lines derived from controllers show enhanced virus suppression when compared with progressors. Virus suppression was mediated in an major histocompatibility complex-dependent manner and found to correlate with a polyfunctional IL-2 CD8 T-cell response. CONCLUSIONS: Using a sensitive in vitro suppression assay, we demonstrate that CD8 T-cell-mediated suppression of HIV-1 replication is a marker of HIV-1 control. Suppressive capacity was found to correlate with polyfunctional IL-2 production. Assessment of CD8 T-cell-mediated suppression may be an important tool to evaluate vaccine-induced responses.

CD8 T cell response and evolutionary pressure to HIV-1 cryptic epitopes derived from antisense transcription.

Retroviruses pack multiple genes into relatively small genomes by encoding several genes in the same genomic region with overlapping reading frames. Both sense and antisense HIV-1 transcripts contain open reading frames for known functional proteins as well as numerous alternative reading frames (ARFs). At least some ARFs have the potential to encode proteins of unknown function, and their antigenic properties can be considered as cryptic epitopes (CEs). To examine the extent of active immune response to virally encoded CEs, we analyzed human leukocyte antigen class I-associated polymorphisms in HIV-1 gag, pol, and nef genes from a large cohort of South Africans with chronic infection. In all, 391 CEs and 168 conventional epitopes were predicted, with the majority (307; 79%) of CEs derived from antisense transcripts. In further evaluation of CD8 T cell responses to a subset of the predicted CEs in patients with primary or chronic infection, both sense- and antisense-encoded CEs were immunogenic at both stages of infection. In addition, CEs often mutated during the first year of infection, which was consistent with immune selection for escape variants. These findings indicate that the HIV-1 genome might encode and deploy a large potential repertoire of unconventional epitopes to enhance vaccine-induced antiviral immunity.

Mass spectrometric investigation of protein alkylation by the RNA footprinting probe kethoxal.

The reactivity of the RNA footprinting reagent kethoxal (KT) toward proteins was investigated by electrospray ionization-Fourier transform mass spectrometry. Using standard peptides, KT was shown to selectively modify the guanidino group of arginine side chains at neutral pH, while primary amino groups of lysine and N-terminus were found to be unreactive under these conditions. Gas-phase fragmentation of KT adducts provided evidence for a cyclic 1,2-diol structure. Esterification of the 1,2-diol product was obtained in borate buffer, and its structure was also investigated by tandem mass spectrometry. When model proteins were probed with this RNA footprinting reagent, the adducts proved to be sufficiently stable to allow for the application of different peptide-mapping procedures to identify the location of modified arginines. Probing of proteins under native folding conditions provided modification patterns that very closely matched the structural context of arginines in the global protein structure. A strong correlation was demonstrated between the susceptibility to modification and residue accessibility calculated from the known 3D structure. When the complexes formed by HIV-1 nucleocapsid (NC) protein and RNA stemloops SL2 and SL3 were investigated, KT footprinting provided accurate information regarding the involvement of individual arginines in binding RNA and showed different reactivity according to their mode of interaction.