Bifunctional degraders, also referred to as proteolysis-targeting chimeras (PROTACs), are a recently developed class of small molecules. They were designed to specifically target endogenous proteins for ubiquitin/proteasome-dependent degradation and to thereby interfere with pathological mechanisms of diseases, including cancer. In this study, we hypothesized that this process of acute pharmacologic protein degradation might increase the direct MHC class I presentation of degraded targets. By studying this question, we contribute to an ongoing discussion about the origin of peptides feeding the MHC class I presentation pathway. Two scenarios have been postulated: peptides can either be derived from homeostatic turnover of mature proteins and/or from short-lived defective ribosomal products (DRiPs), but currently, it is still unclear to what ratio and efficiency both pathways contribute to the overall MHC class I presentation. We therefore generated the intrinsically stable model antigen GFP-S8L-F12 that was susceptible to acute pharmacologic degradation via the previously described degradation tag (dTAG) system. Using different murine cell lines, we show here that the bifunctional molecule dTAG-7 induced rapid proteasome-dependent degradation of GFP-S8L-F12 and simultaneously increased its direct presentation on MHC class I molecules. Using the same model in a doxycycline-inducible setting, we could further show that stable, mature antigen was the major source of peptides presented, thereby excluding a dominant role of DRiPs in our system. This study is, to our knowledge, the first to investigate targeted pharmacologic protein degradation in the context of antigen presentation and our data point toward future applications by strategically combining therapies using bifunctional degraders with their stimulating effect on direct MHC class I presentation.

RNA-dependent RNA polymerases (RdRps) play a key role in the life cycle of RNA viruses and impact their immunobiology. The arenavirus lymphocytic choriomeningitis virus (LCMV) strain Clone 13 provides a benchmark model for studying chronic infection. A major genetic determinant for its ability to persist maps to a single amino acid exchange in the viral L protein, which exhibits RdRp activity, yet its functional consequences remain elusive. To unravel the L protein interactions with the host proteome, we engineered infectious L protein-tagged LCMV virions by reverse genetics. A subsequent mass-spectrometric analysis of L protein pulldowns from infected human cells revealed a comprehensive network of interacting host proteins. The obtained LCMV L protein interactome was bioinformatically integrated with known host protein interactors of RdRps from other RNA viruses, emphasizing interconnected modules of human proteins. Functional characterization of selected interactors highlighted proviral (DDX3X) as well as antiviral (NKRF, TRIM21) host factors. To corroborate these findings, we infected Trim21^-/- mice with LCMV and found impaired virus control in chronic infection. These results provide insights into the complex interactions of the arenavirus LCMV and other viral RdRps with the host proteome and contribute to a better molecular understanding of how chronic viruses interact with their host. RNA-dependent RNA-polymerases (RdRps) play a key role in the life cycle of RNA viruses. They interact with cellular proteins during replication and transcription processes and impact the immunobiology of viral infections. This study characterized the host protein interactome of the RdRp-containing L protein of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV). Several L protein interactors with proviral and antiviral effects were identified in vitro, and mice lacking the identified L protein interactor TRIM21 exhibited impaired control of chronic LCMV infection. Integration of the L protein interactomes with known RdRp interactomes from other RNA viruses highlighted common and virus-specific strategies to interact with the host proteome, which may indicate novel avenues for antiviral interventions.

Immune checkpoint blockade (ICB) immunotherapies have emerged as promising strategies for the treatment of cancer; however, there remains a need to improve their efficacy. Determinants of ICB efficacy are the frequency of tumor mutations, the associated neoantigens, and the T cell response against them. Therefore, it is expected that neoantigen vaccinations that boost the antitumor T cell response would improve ICB therapy efficacy. The aim of this study was to develop a highly immunogenic vaccine using pattern recognition receptor agonists in combination with synthetic long peptides to induce potent neoantigen-specific T cell responses. We determined that the combination of the TLR9 agonist K-type CpG oligodeoxynucleotides (K3 CpG) with the STING agonist c-di-AMP (K3/c-di-AMP combination) significantly increased dendritic cell activation. We found that immunizing mice with 20-mer of either an OVA peptide, low-affinity OVA peptides, or neopeptides identified from mouse melanoma or lung mesothelioma, together with K3/c-di-AMP, induced potent Ag-specific T cell responses. The combined K3/c-di-AMP adjuvant formulation induced 10 times higher T cell responses against neopeptides than the TLR3 agonist polyinosinic:polycytidylic acid, a derivative of which is the leading adjuvant in clinical trials of neoantigen peptide vaccines. Moreover, we demonstrated that our K3/c-di-AMP vaccine formulation with 20-mer OVA peptide was capable of controlling tumor growth and improving survival in B16-F10-OVA tumor-bearing C57BL/6 mice and synergized with anti-PD-1 treatment. Together, our findings demonstrate that the K3/c-di-AMP vaccine formulation induces potent T cell immunity against synthetic long peptides and is a promising candidate to improve neoantigen vaccine platform.

The induction of CTL responses by vaccines is important to combat infectious diseases and cancer. Biodegradable poly(lactic-co-glycolic acid) (PLGA) microspheres and synthetic long peptides are efficiently internalized by professional APCs and prime CTL responses after cross-presentation of Ags on MHC class I molecules. Specifically, they mainly use the cytosolic pathway of cross-presentation that requires endosomal escape, proteasomal processing, and subsequent MHC class I loading of Ags in the endoplasmic reticulum (ER) and/or the endosome. The vesicle SNARE protein Sec22b has been described as important for this pathway by mediating vesical trafficking for the delivery of ER-derived proteins to the endosome. As this function has also been challenged, we investigated the role of Sec22b in cross-presentation of the PLGA microsphere–encapsulated model Ag OVA and a related synthetic long peptide. Using CRISPR/Cas9-mediated genome editing, we generated Sec22b knockouts in two murine C57BL/6-derived APC lines and found no evidence for an essential role of Sec22b. Although pending experimental evidence, the target SNARE protein syntaxin 4 (Stx4) has been suggested to promote cross-presentation by interacting with Sec22b for the fusion of ER-derived vesicles with the endosome. In the current study, we show that, similar to Sec22b, Stx4 knockout in murine APCs had very limited effects on cross-presentation under the conditions tested. This study contributes to characterizing cross-presentation of two promising Ag delivery systems and adds to the discussion about the role of Sec22b/Stx4 in related pathways. Our data point toward SNARE protein redundancy in the cytosolic pathway of cross-presentation.

Meesilpavikkai K, et al. Immune Netw. 2025 Apr;25(2):e18. https://doi.org/10.4110/in.2025.25.e18