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The cell cycle (CC) underpins diverse cell processes like cell differentiation, cell expansion, and tumorigenesis but current single-cell (sc) strategies study CC as: coarse phases, rely on transcriptomic signatures, use imaging modalities limited to adherent cells, or lack high-throughput multiplexing. To solve this, we develop an expanded, Mass Cytometry (MC) approach with 48 CC-related molecules that deeply phenotypes the diversity of scCC states. Using Cytometry by Time of Flight, we quantify scCC states across suspension and adherent cell lines, and stimulated primary human T cells. Our approach captures the diversity of scCC states, including atypical CC states beyond canonical definitions. Pharmacologically-induced CC arrest reveals that perturbations exacerbate noncanonical states and induce previously unobserved states. Notably, primary cells escaping CC inhibition demonstrated aberrant CC states compared to untreated cells. Our approach enables deeper phenotyping of CC biology that generalizes to diverse cell systems with simultaneous multiplexing and integration with MC platforms. Subject terms: Assay systems, Proteomics, Cell biology, Immunology, Systems biology

Reactivation of the latent viral reservoirs is crucial for a cure of HIV/AIDS. However, current latency reversing agents are inefficient, and the endogenous factors that have the potential to reactivate HIV in vivo remain poorly understood. To identify natural activators of latent HIV-1, we screened a comprehensive peptide/protein library derived from human hemofiltrate, representing the entire blood peptidome, using J-Lat cell lines harboring transcriptionally silent HIV-1 GFP reporter viruses. Fractions potently reactivating HIV-1 from latency contained human Retinol Binding Protein 4 (RBP4), the carrier of retinol (Vitamin A). We found that retinol-bound holo-RBP4 but not retinol-free apo-RBP4 strongly reactivates HIV-1 in a variety of latently infected T cell lines. Functional analyses indicate that this reactivation involves activation of the canonical NF-κB pathway and is strengthened by JAK/STAT5 and JNK signalling but does not require retinoic acid production. High levels of RBP4 were detected in plasma from both healthy individuals and people living with HIV-1. Physiological concentrations of RBP4 induced significant viral reactivation in latently infected cells from individuals on long-term antiretroviral therapy with undetectable viral loads. As a potent natural HIV-1 latency-reversing agent, RBP4 offers a novel approach to activating the latent reservoirs and bringing us closer to a cure. Subject terms: Preclinical research, Infectious diseases

HexaBody-CD27 (GEN1053/BNT313) is an investigational novel agonistic CD27 antibody engineered to enhance T-cell costimulation and promote antitumor immunity. Through the introduction of a hexamerization-enhancing mutation in the IgG Fc domain, HexaBody-CD27 was designed to drive clustering and activation of CD27 via intermolecular Fc:Fc interactions between membrane-bound antibodies, independent of crosslinking by FcγR-bearing cells. HexaBody-CD27 carries an Fc-silencing mutation to prevent T-cell depletion through Fc-mediated effector functions. In vitro, HexaBody-CD27 induced CD27 receptor signaling independent of FcγR-mediated crosslinking in a reporter assay. It also enhanced T-cell proliferation, cytotoxic activity and proinflammatory cytokine secretion in primary human lymphocytes. In contrast to benchmark IgG1 CD27 antibodies, HexaBody-CD27 did not induce phagocytosis of T cells in vitro. HexaBody-CD27 promoted ex vivo tumor infiltrating lymphocyte (TIL) expansion in non-small cell lung cancer (NSCLC) specimens, in particular of CD8 + TILs. The combination of HexaBody-CD27 with an anti-PD-1 antibody enhanced T-cell proliferation, cytokine secretion, and cytotoxic activity in vitro compared to either compound alone. In conclusion, HexaBody-CD27 enhanced T-cell activation and effector functions in an FcγR-crosslinking-independent manner, without inducing T-cell depletion. The immune agonist activity of HexaBody-CD27 was potentiated in combination with PD-1 blockade.