InVivoPlus human IgG1 isotype control

Programmed cell death-1 (PD-1) is a co-inhibitory receptor expressed on T cells that dampens TCR and CD28 signaling in the immunological synapse. PD-1 is significantly upregulated on T cells in the tumor microenvironment, where it promotes exhaustion in the context of chronic antigen restimulation. Exhaustion renders T cells hyporesponsive and ineffectual, but potentially resistant to restimulation-induced cell death (RICD). Restimulation-induced cell death (RICD) is a critical propriocidal apoptosis program triggered in activated T cells upon robust TCR re-engagement, which serves to constrain effector T cell expansion and longevity to prevent collateral tissue damage. While the checkpoint function of PD-1 has profound implications for cancer immunotherapy, the role of PD-1 in regulating newly activated T cells remains unclear. We hypothesized that PD-1 attenuates RICD sensitivity in human effector T cells by modulating TCR signal strength. Here we show that transient upregulation of PD-1 helps to protect clonally expanding human CD4+ and CD8 + T cells from premature RICD, with only moderate protection noted in terminally-differentiated, PD-1lo effector CD8 + T cells. Restimulation of T cells with beads containing PD-L1 results in significant apoptosis resistance, dependent on PD-L1 dosage and the proximity of PD-L1 to the TCR and CD28. Interestingly, PD-L1 demonstrated a more significant RICD rescue with CD28 co-ligation as opposed to TCR engagement alone, suggesting PD-1 signaling targets both signaling pathways in this context. Furthermore, biochemical/proteomic data suggest PD-1 modulates proximal signaling downstream of both TCR and CD28 and influences the expression of specific pro/anti-apoptotic proteins that govern RICD sensitivity. Despite the original assumption of PD-1 as a programmed death-inducing protein, our research reveals that homeostatic expression of PD-1 in clonally expanding T cells confers RICD resistance that promotes T cell survival and persistence. These findings present significant implications for understanding how blocking or engaging the PD-L1:PD-1 signaling axis may influence apoptosis sensitivity in both normal and exhausted T cells to alter adaptive immune responses.

Colorectal cancer and liver metastases are a leading cause of cancer-related mortality. Overexpression of the immunostimulatory cytokine TNFSF14/LIGHT associates with improved survival and correlates with increased tumor-infiltrating lymphocytes in patients and a clinically relevant model of colorectal liver metastases. We demonstrate that LIGHT monotherapy activates T cells, but also induces T cell exhaustion and the recruitment of immunosuppressive elements. As colorectal liver metastases exhibit high levels of CTLA-4 expression, we combined LIGHT overexpression with anti-CTLA-4, leading to complete tumor control. The combination functions by homing tumor-infiltrating lymphocytes, inducing tumor antigen-specific T cells, and reversing T cell exhaustion. Whereas both LIGHT overexpression and anti-CTLA-4 increase tumor-promoting macrophages, the combination eliminates this population. The ability of LIGHT overexpression combined with CTLA-4 inhibition to reverse T cell exhaustion and myeloid cell suppression is supported by analysis of complementary patient cohorts and has strong clinical relevance, especially given that liver metastases contribute to immunotherapy resistance across various cancer types.

Sjögren's disease (SjD) is an autoimmune disease that causes salivary gland dysfunction due to immune-mediated destruction. While autoantibodies such as anti-SSA and anti-centromere (CENT) are associated with distinct clinical manifestations, the molecular features remain to be elucidated. In this study, we apply multi-modal single-cell technologies: single-cell RNA sequencing, T cell and B cell receptor sequencing and spatial transcriptomics to salivary gland lesions, aiming to elucidate common and unique cellular and transcriptional signatures linked to different autoantibody profiles. Our analysis demonstrates that GZMB+GNLY+ CD8+ T cells are the main expanded subset across different autoantibody statuses, highlighting their central role in SjD pathogenesis, while the enrichment of memory B cells is more prominent in anti-CENT-positive patients. Cytokine signaling also differs by autoantibody profile, with an activated interferon signature in anti-SSA-positive patients, whereas TGFβ signaling is enhanced in anti-CENT-positive patients. Furthermore, spatial profiling reveals THY1+ fibroblasts, expressing complement genes and chemokines, as key hubs orchestrating inflammation within the salivary glands. These findings deepen our understanding of the pathogenesis of SjD, and may inform the development of targeted and personalized therapeutic strategies.

There is intense interest in the advancement of RNAs as rationally designed therapeutic agents, especially in oncology, where a major focus is to use RNAs to stimulate pattern recognition receptors to leverage innate immune responses. However, the inability to selectively deliver therapeutic RNAs within target cells after intravenous administration now hinders the development of this type of treatment for cancer and other disorders. Here, we found that a tumor-targeting, cell-penetrating, and RNA binding monoclonal antibody, TMAB3, can form stable, noncovalent antibody/RNA complexes of a discrete size that mediate highly specific and functional delivery of RNAs into tumors. Using 3p-hpRNA, an agonist of the pattern recognition receptor retinoic acid-inducible gene-I (RIG-I), we observed robust antitumor efficacy of systemically administered TMAB3/3p-hpRNA complexes in mouse models of pancreatic cancer, medulloblastoma, and melanoma. In the KPC syngeneic, orthotopic pancreatic cancer model in immunocompetent mice, treatment with TMAB3/3p-hpRNA tripled animal survival, decreased tumor growth, and specifically targeted malignant cells, with a 1500-fold difference in RNA delivery into tumor cells versus nonmalignant cells within the tumor mass. Single-cell RNA sequencing (scRNA-seq) and flow cytometry demonstrated that TMAB3/3p-hpRNA treatment elicited a potent antitumoral immune response characterized by RIG-I activation and increased infiltration and activity of cytotoxic T cells. These studies established that TMAB3/RNA complexes can deliver RNA payloads specifically to hard-to-treat tumor cells to achieve antitumor efficacy, providing an antibody-based platform to advance the study of RNA therapies for the treatment of patients with cancer.