The immune system’s ability to recognize peptides on major histocompatibility molecules (pMHCs) contributes to eradication of cancers and pathogens. Tracking these responses in vivo could help evaluate the efficacy of immune interventions and improve mechanistic understanding of immune responses. We employ synTacs, dimeric pMHC scaffolds of defined composition, which enable clonal-selective delivery of a variety of signaling, recruitment, and imaging modalities. We show that synTacs, when labeled with positron-emitting isotopes, can non-invasively image antigen-specific CD8 T cells in vivo . We imaged human papillomavirus (HPV16) E7-specific CD8 T cells by positron emission tomography with an HPV16 E7 peptide-loaded synTac in HPV16-positive tumors, following administration of a therapeutic vaccine. We also imaged influenza A virus (IAV) nucleoprotein-specific CD8 T cells in the lungs of IAV-infected mice, using an isotopically labeled flu-specific synTac. It is thus possible to visualize antigen-specific CD8 T cell populations in vivo , which may serve prognostic and diagnostic roles.

A detailed understanding of the energetic contributions to histone peptide recognition would be valuable for a better understanding of chromatin anchoring mechanisms and histone diagnostic design. Here, we probed the energetic contributions to recognize the same unmodified histone H3 by three different plant homeodomain (PHD) H3K4me0 readers: hKDM5B-PHD1 (first PHD finger of hKDM5B), hBAZ2A-PHD, and hAIRE-PHD1. The energetic contributions of residues differ significantly from one complex to the next. For example, H3K4A substitution completely aborts the formation of the hAIRE-histone peptide complex, while it has only a small destabilizing effect on binding of the other readers, even though H3K4 methylation disrupts all three complexes. Packing density suggests that methylation of more tightly packed Lys/Arg residues can disrupt binding, even if the energetic contribution is small. The binding behavior of hKDM5B-PHD1 and hBAZ2A-PHD is similar, and like PHD H3R2 readers, both possess a pair of Asp residues in the treble clef for interaction with H3R2. PHD subtype sequences, especially the tandem PHD-PHD fingers, show enrichment in the treble clef Asp residues, suggesting that it is a subtype-specific property. These Asp residues make significant energetic contributions to the formation of the hKDM5B-histone peptide complex, suggesting that there are interactions in addition to those reported in the recent NMR structure. However, the presence of the treble clef Asp in PHD sequences may not always be sufficient for histone peptide binding. This study showcases reader-histone peptide interactions in the context of residue conservation, energetic contributions, interfacial packing, and sequence-based reader subtype predictability.

BackgroundImmunotherapies are highly promising and effective strategies for the treatment of cancer; however, continuing challenges persist, including 1) untargeted global immune modulation, resulting in serious side effects; 2) lack of therapeutics capable of in vivo expansion of tumor-specific T cells; 3) inability to visualize in vivo tumor-specific T cell responses; and 4) lack of flexible platforms to rapidly and efficiently explore new therapeutic strategies and immune-escape mechanisms. To address these challenges, we developed a novel class of precision biologics to treat cancer, autoimmune diseases and infectious diseases. We describe a modular platform constructed around an Fc-based covalent pMHC dimer, referred to as synTac (artificial synapse for T cell activation; also termed Immuno-STATs for Selective Targeting and Alteration of T cells), which selectively delivers different cargoes, including costimulatory, coinhibitory or cytokine signals and other modalities to primary T cells of defined specificity. The inherent modularity supports broad applications. Changing the encoded peptide enables targeting of different T cell specificities to address different diseases, while altering the cargo allows for evaluation of different co-modulatory mechanisms or the delivery of mechanistically informative probes.MethodsSortase A-mediated enzymatic coupling supported site-specific and stoichiometric installation of positron emission tomography (PET)-active radiolabels on synTacs to visualize the in vivo localization of antigen-specific T cells. The NSG humanized mouse model allowed for the evaluation of synTacs/Immuno-STATs to drive the in vivo antigen-specific expansion of human CD8 T cells.ResultsUsing radiolabeled synTacs/Immuno-STATs loaded with the appropriate peptides, we employed positron emission tomography to localize human papillomavirus (HPV16)-specific CD8 T cells to implanted HPV16-positive tumors in mice, as well as influenza A virus (IAV)-specific CD8 T cells in the lungs of IAV-infected mice. In vivo administration of HIV- and CMV-specific synTacs/Immuno-STATs to immunodeficient mice intrasplenically engrafted with human donor PBMCs resulted in the marked and selective expansion of HIV-specific and CMV-specific human CD8 T cells populating their spleens, respectively.ConclusionsWe demonstrate the remarkable flexibility of the synTacs/Immuno-STAT platform for addressing a broad range of applications, including the first report of the in vivo imaging of antigen-specific CD8 T cell populations and in vivo antigen-selective expansion of human CD8 T cells. These results suggest that, in addition to broad therapeutic applications, synTac/Immuno-STATs may provide prognostic/diagnostic information. Most notably, these results demonstrate the presence of synTacs/Immuno-STAT biologics in the tumor or infected tissues where they can elicit T cell restimulation and expansion necessary for target killing and enhanced therapeutic efficacy.