HLA-E-restricted HIV-1-specific CD8+ T cell responses in natural infection.

CD8+ T cell responses restricted by MHC-E, a nonclassical MHC molecule, have been associated with protection in an SIV/rhesus macaque model. The biological relevance of HLA-E-restricted CD8+ T cell responses in HIV infection, however, remains unknown. In this study, CD8+ T cells responding to HIV-1 Gag peptides presented by HLA-E were analyzed. Using in vitro assays, we observed HLA-E-restricted T cell responses to what we believe to be a newly identified subdominant Gag-KL9 as well as a well-described immunodominant Gag-KF11 epitope in T cell lines derived from chronically HIV-infected patients and also primed from healthy donors. Blocking of the HLA-E/KF11 binding by the B7 signal peptide resulted in decreased CD8+ T cell responses. KF11 presented via HLA-E in HIV-infected cells was recognized by antigen-specific CD8+ T cells. Importantly, bulk CD8+ T cells obtained from HIV-infected individuals recognized infected cells via HLA-E presentation. Ex vivo analyses at the epitope level showed a higher responder frequency of HLA-E-restricted responses to KF11 compared with KL9. Taken together, our findings of HLA-E-restricted HIV-specific immune responses offer intriguing and possibly paradigm-shifting insights into factors that contribute to the immunodominance of CD8+ T cell responses in HIV infection.

Phosphorylated Gß is a directional cue during yeast gradient tracking.

Budding yeast cells interpret shallow pheromone gradients from cells of the opposite mating type, polarize their growth toward the pheromone source, and fuse at the chemotropic growth site. We previously proposed a deterministic, gradient-sensing model that explains how yeast cells switch from the intrinsically positioned default polarity site (DS) to the gradient-aligned chemotropic site (CS) at the plasma membrane. Because phosphorylation of the mating-specific Gß subunit is thought to be important for this process, we developed a biosensor that bound to phosphorylated but not unphosphorylated Gß and monitored its spatiotemporal dynamics to test key predictions of our gradient-sensing model. In mating cells, the biosensor colocalized with both Gß and receptor reporters at the DS and then tracked with them to the CS. The biosensor concentrated on the leading side of the tracking Gß and receptor peaks and was the first to arrive and stop tracking at the CS. Our data showed that the concentrated localization of phosphorylated Gß correlated with the tracking direction and final position of the G protein and receptor, consistent with the idea that gradient-regulated phosphorylation and dephosphorylation of Gß contributes to gradient sensing. Cells expressing a nonphosphorylatable mutant form of Gß exhibited defects in gradient tracking, orientation toward mating partners, and mating efficiency.

A Recombinant Affinity Reagent Specific for a Phosphoepitope of Akt1.

The serine/threonine-protein kinase, Akt1, plays an important part in mammalian cell growth, proliferation, migration and angiogenesis, and becomes activated through phosphorylation. To monitor phosphorylation of threonine 308 in Akt1, we developed a recombinant phosphothreonine-binding domain (pTBD) that is highly selective for the Akt1 phosphopeptide. A phage-display library of variants of the Forkhead-associated 1 (FHA1) domain of yeast Rad53p was screened by affinity selection to the phosphopeptide, 301-KDGATMKpTFCGTPEY-315, and yielded 12 binding clones. The strongest binders have equilibrium dissociation constants of 160⁻180 nanomolar and are phosphothreonine-specific in binding. The specificity of one Akt1-pTBD was compared to commercially available polyclonal antibodies (pAbs) generated against the same phosphopeptide. The Akt1-pTBD was either equal to or better than three pAbs in detecting the Akt1 pT308 phosphopeptide in ELISAs.

Generating a recombinant phosphothreonine-binding domain for a phosphopeptide of the human transcription factor, c-Myc.

Transcription factor c-Myc is an oncoprotein that is regulated at the post-translational level through phosphorylation of two conserved residues, Serine 62 (Ser62) and Threonine 58 (Thr58). A highly specific tool capable of recognizing Myc via pThr58 is needed to monitor activation and localization. Through phage display, we have isolated 10 engineered Forkhead-associated (FHA) domains that selectively bind to a phosphothreonine (pThr)-containing peptide (53-FELLPpTPPLSPS-64) segment of human c-Myc. One domain variant was observed to bind to the Myc-pThr58 peptide with a KD value of 800 nM and had >1000-fold discrimination between the phosphorylated and non-phosphorylated peptide. The crystal structure of the engineered FHA Myc-pThr-binding domain (Myc-pTBD) was solved in complex with its cognate ligand. The Myc-pTBD was observed to be structurally similar to the yeast Rad9 FHA1 domain, except that its ß4-ß5 and ß10-ß11 loops form a hydrophobic pocket to facilitate the interaction between the domain and the peptide ligand. The Myc-pTBD's specificity for its cognate ligand was demonstrated to be on a par with 3 commercial polyclonal antibodies, suggesting that this recombinant reagent is a viable alternative to antibodies for monitoring Myc regulation.

A comparison of phosphospecific affinity reagents reveals the utility of recombinant Forkhead-associated domains in recognizing phosphothreonine-containing peptides.

Phosphorylation is an important post-translational event that has a wide array of functional consequences. With advances in the ability of various technologies in revealing and mapping new phosphosites in proteins, it is equally important to develop affinity reagents that can monitor such post-translational modifications in eukaryotic cells. While monoclonal and polyclonal antibodies have been shown to be useful in assessing the phosphoproteome, we have expanded our efforts to exploit the Forkhead-associated 1 (FHA1) domain as scaffold for generating recombinant affinity reagents that recognize phosphothreonine-containing peptides. A phage display library of FHA1 variants was screened by affinity selection with 15 phosphothreonine-containing peptides corresponding to various human transcription factors and kinases, including human Myc, calmodulin-dependent protein kinase II (CaMKII), and extracellular-signal regulated kinases 1 and 2 (ERK1/2). The library yielded binding variants against 10 targets (66% success rate); success was largely determined by what residue occurred at the +3 position (C-terminal) to the pThr moiety (i.e., pT+3). The FHA domains binding Myc, CaMKII, and ERK1/2 were characterized and compared against commercially available antibodies. All FHA domains were shown to be phosphorylation-dependent and phosphothreonine-specific in their binding, unlike several commercial monoclonal and polyclonal antibodies. Both the pThr and the residue at the pT+3 position were major factors in defining the specificity of the FHA domains.

The nucleocapsid protein of Rift Valley fever virus is a potent human CD8+ T cell antigen and elicits memory responses.

There is no licensed human vaccine currently available for Rift Valley Fever Virus (RVFV), a Category A high priority pathogen and a serious zoonotic threat. While neutralizing antibodies targeting the viral glycoproteins are protective, they appear late in the course of infection, and may not be induced in time to prevent a natural or bioterrorism-induced outbreak. Here we examined the immunogenicity of RVFV nucleocapsid (N) protein as a CD8(+) T cell antigen with the potential for inducing rapid protection after vaccination. HLA-A*0201 (A2)-restricted epitopic determinants were identified with N-specific CD8(+) T cells from eight healthy donors that were primed with dendritic cells transduced to express N, and subsequently expanded in vitro by weekly re-stimulations with monocytes pulsed with 59 15mer overlapping peptides (OLPs) across N. Two immunodominant epitopes, VT9 (VLSEWLPVT, N(121-129)) and IL9 (ILDAHSLYL, N165-173), were defined. VT9- and IL9-specific CD8(+) T cells identified by tetramer staining were cytotoxic and polyfunctional, characteristics deemed important for viral control in vivo. These peptides induced specific CD8(+) T cell responses in A2-transgenic mice, and more importantly, potent N-specific CD8(+) T cell reactivities, including VT9- and IL9-specific ones, were mounted by mice after a booster vaccination with the live attenuated RVF MP-12. Our data suggest that the RVFV N protein is a potent human T cell immunogen capable of eliciting broad, immunodominant CD8(+) T cell responses that are potentially protective. Understanding the immune responses to the nucleocapsid is central to the design of an effective RVFV vaccine irrespective of whether this viral protein is effective as a stand-alone immunogen or only in combination with other RVFV antigens.