Foreign and self-antigens activate CD4+ conventional and regulatory T cells (Tregs) to promote immunity and tolerance, respectively. These cell populations, which depend on interleukin-2 (IL-2), are being expanded and engineered in vitro for adoptive cell therapy (ACT) for cancer and autoimmunity. Here, we investigate the molecular pathways underlying the in vitro expansion of human CD4+ Teff and Tregs to TCR/CD28/IL-2 signaling over 12-days. Temporal integration of differential chromatin accessibility and gene expression revealed similar responses over the first 6 days. After this time, T effector (Teff) cells showed greater expansion that was associated with more robust gene activation and chromatin opening that supported increased activation of mTORC1-dependent signaling and a more energetic phenotype. Thus, Tregs are programmed temporally for more limited expansion in vitro that may benefit ACT for cancer but may be a drawback for autoimmunity. These findings may reflect a mechanism to finely tune Treg numbers to maintain homeostasis in vivo.
© 2025 The Authors.

The challenges that remain in the treatment of solid tumors with chimeric antigen receptor (CAR)-T cells include limited solid tumor-specific targets and poor CAR-T cell expansion and function due to limited availability of solid tumor antigens outside the tumor microenvironment. Prostate cancer is the second most common cancer among men worldwide. Current CAR-T therapies for prostate cancer lack specific targets, posing safety risks. To overcome these problems, we identified prostatic acid phosphatase (PAP, also known as ACPP or ACP3) as a feasible CAR-T target for prostate cancer and developed CoupledCAR, a novel approach for expanding tumor-targeting CAR-T cells without tumor antigens.
We analyzed the expression of PAP from The Cancer Genome Atlas database and validated its expression in normal and cancer tissues through immunohistochemistry staining. To generate anti-PAP specific antibodies, we screened the human single-chain antibody library using transmembrane PAP-His antigen and selected antibodies based on their binding ability and specificity. We constructed PAP-targeted CAR and evaluated their antitumor efficacy both in vitro and in vivo. We validated the function of PAP CoupledCAR in both in vitro and in vivo experiments, and further analyzed its mechanism using single-cell RNA sequencing (scRNA-Seq).
PAP was specifically expressed in prostate epithelial and prostate cancer cells, with no expression in other tissues. Seven single-chain variable fragments were screened from the human single-chain antibody library, with S5D1 showing the highest binding ability for the PAP. PAP CAR-T cells demonstrated strong antitumor efficacy both in vitro and in vivo. Furthermore, the CoupledCAR system significantly expanded PAP CAR-T cells, promoting memory-like status, reducing exhaustion, and enhancing their antitumor efficacy. The scRNA-Seq demonstrated that the expansion of PAP CAR-T cells in the CoupledCAR system is mediated by costimulatory signals and cytokine signals, rather than T-cell receptor signals.
Our study is the first to demonstrate that PAP is a specific target for CAR-T therapy in prostate cancer, both in vitro and in vivo. We developed the CoupledCAR platform technology for solid tumor CAR-T cell therapy, enabling the expansion of tumor-targeting CAR-T cells without requiring tumor antigens and thereby enhancing their functionality against solid tumors.
© Author(s) (or their employer(s)) 2025. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ Group.

Within the bone marrow (BM), the intercellular communication between hematopoietic stem and progenitor cells (HSPCs) and mesenchymal stem/stromal cells (MSCs) is critical for the life-long maintenance of functional hematopoiesis. In recent years, the transfer of mitochondria between MSCs and HSPCs has emerged as a key aspect of this communication, occurring both in stress and homeostatic conditions. In human, the mesenchymal-to-hematopoietic transfer process and functional impact remain cryptic, primarily due to a lack of robust models. To this end, we here describe the development and exploitation of iMSOD-mito, an immortalized human MSCs line bearing an inducible mCherry mitochondrial tag. Co-culture with primary healthy HSPCs or a leukemic cell line revealed a high mitochondrial transfer rate (>15%), exclusively relying on cell-to-cell contact. While all CD34+ blood cells received mitochondria, a preferential transfer towards phenotypic hematopoietic stem cells was identified. Similarly, using primary MSCs with genetically labelled mitochondria we confirmed a transfer to all CD34+ populations, albeit occurring at a lower frequency than with the iMSOD-mito (3.38%). By engineering 3D bone marrow niches in perfusion bioreactor, this transfer rate could be significantly increased, while the biased towards HSC as receiver was maintained. Functionally, mitochondria-receiving cells exhibited an increased mitochondria membrane potential and reactive oxygen species (ROS) production, which in HSPCs was associated with retained quiescence in single cell divisional assay. In summary, we propose the iMSOD-mito as a standardized tool to model human mesenchymal-to-hematopoietic mitochondria transfer in 2D or 3D culture systems. Our work prompts the study of mitochondria transfer in both healthy or disease conditions, towards the design of regenerative therapies or identification of new targets in a malignant context.

Mutations in the ELANE gene, encoding the neutrophil elastase (NE) protein, are responsible for most cyclic neutropenia (CyN) cases and approximately 25% of congenital neutropenia (CN) cases. In CN and in CyN, a median of 2.8% of CD34+ cells were early CD49f+ hematopoietic stem cells (eHSC) that did not express ELANE and thus escape from the unfolded protein response (UPR) caused by mutated NE. In CyN, the CD49f+ cells respond to granulocyte colony-stimulating factor (G-CSF) with a significant upregulation of the hematopoietic stem cell-specific transcription factors, C/EBPα, MLL1, HOXA9, MEIS1, and HLF during the ascending arm of the cycle, resulting in the differentiation of myeloid cells to mature neutrophils at the cycle peak. However, NE protein released by neutrophils at the cycle's peak caused a negative feedback loop on granulopoiesis through the proteolytic digestion of G-CSF. In contrast, in CN patients, CD49f+ cells failed to express mRNA levels of HSC-specific transcription factors mentioned above. Rescue of C/EBPα expression in CN restored granulopoiesis.

Immunosenescence occurs as people age, leading to an increased incidence of age-related diseases. The number of senescent T cells also rises with age. T cell senescence and immune response dysfunction can result in a decline in immune function, especially in anti-tumor immune responses. Metformin has been shown to have various beneficial effects on health, such as lowering blood sugar levels, reducing the risk of cancer development, and slowing down the aging process. However, the immunomodulatory effects of metformin on senescent T cells still need to be investigated.
PBMCs isolation from different age population (n = 88); Flow Cytometry is applied to determine the phenotypic characterization of senescent T lymphocytes; intracellular staining is applied to determine the function of senescent T cells; Enzyme-Linked Immunosorbent Assay (ELISA) is employed to test the telomerase concentration. The RNA-seq analysis of gene expression associated with T cell senescence.
The middle-aged group had the highest proportion of senescent T cells. We found that metformin could decrease the number of CD8 + senescent T cells. Metformin affects the secretion of SASP, inhibiting the secretion of IFN-γ in CD8 + senescent T cells. Furthermore, metformin treatment restrained the production of the proinflammatory cytokine IL-6 in lymphocytes. Metformin had minimal effects on Granzyme B secretion in senescent T cells, but it promoted the production of TNF-α in senescent T cells. Additionally, metformin increased the concentration of telomerase and the frequency of undifferentiated T cells. The results of RNA-seq showed that metformin promoted the expression of genes related to stemness and telomerase activity, while inhibiting the expression of DNA damage-associated genes.
Our findings reveal that metformin could inhibit T cell senescence in terms of cell number, effector function, telomerase content and gene expression in middle-aged individuals, which may serve as a promising approach for preventing age-related diseases in this population.
© 2023. The Author(s).