Although transplantation is the preferred treatment for end-stage organ disease, long-term outcomes are limited by immunosuppressive drug toxicity and immune-mediated injury. Selective in vivo expansion of regulatory T cells (Tregs) using interleukin-2 (IL-2) analogs has emerged as a strategy to induce antigen-specific transplant tolerance with fewer side effects. Herein, we investigate the therapeutic efficacy of an IL-2 mutein molecule (mIL-2) with enhanced receptor specificity and extended half-life in murine models of solid organ transplantation. mIL-2 therapy significantly improves allograft survival in an antigen-specific manner, accompanied by increased Treg activation, decreased effector T cell activation and reduced donor-specific antibody production. Transcriptional profiling reveals expansion of Tregs expressing the suppression of tumorigenicity 2 (ST2) Tregs with heightened activation status and suppressive function. Accordingly, the mIL-2-induced long-term allograft survival is abrogated in Treg-specific ST2 knockout graft recipients, underscoring the critical role of ST2+ Tregs. These findings identify mIL-2 as an approach to promote long-term transplant tolerance while reducing reliance on conventional immunosuppression.

INTRODUCTION: Triple-negative breast cancer (TNBC) is characterized as the most unfavorable prognosis of the breast cancer subtype. Chemotherapy is currently the primary treatment owing to a persistent lack of effective alternative medicine. To fulfill this unaddressed clinical requirement, we utilized the PGLU-Fc-III-4C MsAb platform to develop a nanobioconjugate trispecific antibody (TsAb; CD3×CD137×CD276) that targeted CD3, CD137, and CD276, aiming to restrict the growth of TNBC tumors (4T1 model) and provide a novel therapeutic strategy. METHODS: The capacity for binding to target cells and anti-tumor effects of TsAb in vitro were evaluated. In vivo antitumor efficacy and biosafety were further assessed in the 4T1 subcutaneous tumor model. Anti-tumor immune responses induced by TsAb on tumor-infiltrating CD8+ T cells were monitored. RESULTS: The TsAb effectively bound and facilitated interactions between 4T1 tumor cells and T cells, significantly boosting the anti-tumor effect. TsAb promoted the expansion of tumor and splenic T lymphocytes and facilitated the recruitment of splenic and blood T lymphocytes to tumor tissues. Compared with the PGLU-Fc-III-4C-IgG treatment group, the TsAb treatment group had varying degrees of increase in CD8+ tissue-resident memory T cells (TRM), central memory T cells (TCM), and terminal effector memory T cells (TEM) in tumor tissues. The TsAb treatment group exhibited a significant increase of PD-1+ CD8+ T cells and TCF1-Tim-3+ CD8+ terminally exhausted T cells in tumor tissues. The safety profile demonstrated no obvious systemic toxicity. DISCUSSION: Briefly, TsAb mediated CD8+ T cell activation, proliferation, and terminal differentiation, accompanied by increasing cytokine production to eliminate the tumor. Meanwhile, no obvious systemic toxicity was observed. In general, the CD3×CD137×CD276 nanobioconjugate trispecific antibody provides a promising immunotherapeutic approach via regulating CD8+ T cell immune response for the treatment of triple-negative breast cancer.

Rapid and uniform labeling of plasma membrane proteins is essential for high-resolution imaging of dynamic membrane topologies and intercellular communication in live mammalian cells. Existing strategies for labeling live cell membranes, such as fluorescent fusion proteins, enzyme-mediated tags, metabolic bioorthogonal labeling, and lipophilic dyes, face trade-offs in the requirement of genetic manipulation, the presence of non-uniform labeling, the need for extensive preparation times, and limited choices of fluorophores. Here, we present a streamlined protocol that leverages N-hydroxysuccinimide (NHS)-ester chemistry to achieve rapid (≤5 min), covalent conjugation of synthetic small-molecule dyes to surface-exposed primary amines, enabling pan-membrane-protein labeling. This workflow covers dye stock preparation, labeling for suspension and adherent cells, multiplex live-cell imaging, fusion protein co-staining (including insulin-triggered receptor endocytosis), 3D membrane visualization, and in vivo assays for visualizing membrane-derived material transfers between donor and recipient cells using a lymphoma T-cell mouse model. This high-density labeling approach is compatible with various cell types across diverse imaging platforms. Its speed, versatility, and stability make it a broadly applicable tool for studying plasma membrane dynamics and intercellular membrane trafficking. Key features • Rapid high-density membrane labeling with small-molecule fluorescent dyes.• Enables live-cell multiplexed imaging, amenable to primary cells and cells expressing fluorescent fusion proteins, and supports in vivo studies of membrane-associated cell–cell communications.• Compatible with various fluorescence imaging modalities.