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Reduced serum testosterone (T),or hypogonadism,affects millions of men and is associated with many pathologies,including infertility,cardiovascular diseases,metabolic syndrome,and decreased libido and sexual function. Administering T-replacement therapy (TRT) reverses many of the symptoms associated with low T levels. However,TRT is linked to side effects such as infertility and increased risk of prostate cancer and cardiovascular diseases. Thus,there is a need to obtain T-producing cells that could be used to treat hypogonadism via transplantation and reestablishment of T-producing cell lineages in the body. T is synthesized by Leydig cells (LCs),proposed to derive from mesenchymal cells of mesonephric origin. Although mesenchymal cells have been successfully induced into LCs,the limited source and possible trauma to donors hinders their application to clinical therapies. Alternatively,human induced pluripotent stem cells (hiPSCs),which are expandable in culture and have the potential to differentiate into all somatic cell types,have become the emerging source of autologous cell therapies. We have successfully induced the differentiation of hiPSCs into either human Leydig-like (hLLCs) or adrenal-like cells (hALCs) using chemically defined culture conditions. Factors critical for the development of LCs were added to both culture systems. hLLCs expressed all steroidogenic genes and proteins important for T biosynthesis,synthesized T rather than cortisol,secreted steroid hormones in response to dibutyryl-cAMP and 22(R)-hydroxycholesterol,and displayed ultrastructural features resembling LCs. By contrast,hALCs synthesized cortisol rather than T. The success in generating hiPSC-derived hLLCs with broad human LC (hLC) features supports the potential for hiPSC-based hLC regeneration. View Publication

Activating interferon responses with STING agonists (STINGa) is a current cancer immunotherapy strategy,and therapeutic modalities that enable tumor-targeted delivery via systemic administration could be beneficial. Here we demonstrate that tumor cell-directed STING agonist antibody-drug-conjugates (STINGa ADCs) activate STING in tumor cells and myeloid cells and induce anti-tumor innate immune responses in in vitro,in vivo (in female mice),and ex vivo tumor models. We show that the tumor cell-directed STINGa ADCs are internalized into myeloid cells by Fcγ-receptor-I in a tumor antigen-dependent manner. Systemic administration of STINGa ADCs in mice leads to STING activation in tumors,with increased anti-tumor activity and reduced serum cytokine elevations compared to a free STING agonist. Furthermore,STINGa ADCs induce type III interferons,which contribute to the anti-tumor activity by upregulating type I interferon and other key chemokines/cytokines. These findings reveal an important role for type III interferons in the anti-tumor activity elicited by STING agonism and provide rationale for the clinical development of tumor cell-directed STINGa ADCs. Activation of the STING pathway can promote anti-tumor immunity. Here the authors generate tumor cell-directed STING agonist antibody-drug conjugates that activate STING in tumor and myeloid cells,promoting anti-tumor innate immune responses in preclinical cancer models. View Publication

Although chimeric antigen receptor (CAR) T cells are effective against B-lineage malignancies,post-CAR relapse is common,and efficacy in other tumors is limited. These challenges may be addressed through rational manipulations to control CAR T cell function. Here we examine the impact of cognate T cell antigen experience on subsequent CD8+ CAR T cell activity. Prior antigen encounter resulted in superior effector function against leukemia expressing low target antigen density at the expense of reduced proliferative capacity and susceptibility to dysfunction at limiting CAR doses. Distinctive temporal transcriptomic and epigenetic profiles in naive-derived and memory-derived CAR T cells identified RUNX family transcription factors as potential targets to augment the function of naive-derived CD8+ CAR T cells. RUNX2 overexpression enhanced antitumor efficacy of mouse CAR T cells,dependent on prior cell state,and heightened human CAR T cell functions. Our data demonstrate that prior antigen experience of CAR T cells determines functional attributes and amenability to transcription factor-mediated functional enhancement. Here,Fry and colleagues examine the impact of antigen experience on subsequent CD8+ CAR T cell activity during the antileukemia response and show that RUNX2 overexpression enhances antitumor activity of these cells. View Publication

IntroductionT cells are major components of the immune system. Their activation requires interaction between the T cell receptor and co-stimulatory molecules,crucial during infection,inflammation,and allogeneic rejection. Monomeric CRP (mCRP) is a known modulator of inflammation and particularly the innate immune response,however its interaction with T cells as part of the adaptive immune response remains unclear.MethodsPeripheral blood mononuclear cells (PBMC) and T cells were isolated. Flow cytometric analysis was conducted to evaluate Fcγ receptor CD16 expression on T cells,the binding of CRP to T cells,and its impact on proliferation and apoptosis. T cell activation was assessed after 1,2,3,5 and 7 days by assessing CD69 and CD25 expression,and under various conditions including coculture with monocytes and several inhibitory factors.ResultsT cells express CD16 that binds mCRP in a concentration-dependent manner,and particularly on activated T cells. While mCRP reduces apoptosis and accelerates proliferation in T cells,it does not independently activate them. However,activation of monocytes by mCRP leads to T cell activation,indicating a direct cell to cell interaction during CRP-induced activation. This effect could be alleviated by inhibition of the CD80/CD28 pathway.ConclusionCRP does not activate T Cells directly but via PI3-kinase-dependent activation of monocytes and subsequent CD80/CD28 cell to cell contact. The findings suggest the effects of CRP on T cells depend on their environment and the presence of other proinflammatory agents. View Publication

Hantaviruses are zoonotically transmitted from rodents to humans through the respiratory route,with no currently approved antivirals or widely available vaccines. The recent discovery of interhuman-transmitted Andes virus (ANDV) necessitates the systematic identification of cell tropism,infective potential,and potent therapeutic agents. We utilized human primary lung endothelial cells,various pluripotent stem cell-derived heart and brain cell types,and established human lung organoid models to evaluate the tropisms of Old World Hantaan (HTNV) and New World ANDV and Sin Nombre (SNV) viruses. ANDV exhibited broad tropism for all cell types assessed. SNV readily infected pulmonary endothelial cells,while HTNV robustly amplified in endothelial cells,cardiomyocytes,and astrocytes. We also provide the first evidence of hantaviral infection in human 3D distal lung organoids,which effectively modeled these differential tropisms. ANDV infection transcriptionally promoted cell injury and inflammatory responses,and downregulated lipid metabolic pathways in lung epithelial cells. Evaluation of selected drug candidates and pharmacotranscriptomics revealed that the host-directed small molecule compound urolithin B inhibited ANDV infection and restored cellular metabolism with minimal changes in host transcription. Given the scarcity of academic BSL-4 facilities that enable in vivo hantaviral studies,this investigation presents advanced human cell-based model systems that closely recapitulate host cell tropism and responses to infection,thereby providing critical platforms to evaluate potential antiviral drug candidates. Author summaryHantaviruses are fatal human pathogens that cause hemorrhagic fevers and are classified into either Old World or New World groups. Though most hantaviruses utilize zoonotic transmission,the New World Andes virus (ANDV) is unique in its ability to spread between humans. This distinct transmission mode underscores the need to investigate its cell tropism,pathogenicity,and therapeutic targets. Thus,we performed a systems-level comparison of the Old World Hantaan virus (HTNV) and New World hantaviruses,ANDV and Sin Nombre virus (SNV),using human lung,heart,and brain cell models,alongside lipidomic and transcriptomic profiling. We observed that ANDV exhibits broad tropism,infecting all tested cell types,including lung epithelial cells. HTNV replicated in lung endothelial,heart,and brain cells,whereas SNV replication was largely confined to lung endothelial cells. Notably,ANDV infection induced stronger host transcriptional changes,promoted cell injury and inflammatory responses,and suppressed lipid metabolic pathways in lung epithelial cells. Further drug testing and pharmacotranscriptomic analysis identified effective inhibitors of ANDV infection,including urolithin B,that restored cellular metabolism with minimal transcriptional disruption. This study provides a comparative framework for understanding hantavirus cell tropism and host responses and highlights potential antiviral candidates for treating these severe viral infections. View Publication

IntroductionAmid the persistent threat of future pandemics,the continuous evolution of SARS-CoV-2 exposed critical challenges for vaccine efficacy and therapeutic interventions,highlighting the need for rapid and adaptable approaches to respond to immune escape variants.MethodsHere,we report the use of immortalized B cell libraries from human peripheral blood mononuclear cells (PBMCs) and tonsil tissues to uncover B cell clones exhibiting cross-reactive neutralization against various SARS-CoV-2 variants and perform directed evolution of immortalized B cell clones to produce antibodies with improved binding and neutralization against emerging SARS-CoV-2 variants.ResultsImmortalization of PBMC and tonsil-derived human B cells was achieved through transduction with retroviral vectors encoding apoptosis inhibitors,yielding transduction efficiencies of 67.5% for PBMCs and 50.2% for tonsil-derived cells. Analysis revealed that immortalized B cell libraries produced with this method retain diverse immunoglobulin isotype representations. Through high-throughput functional screening of approximately 40,000 B cells per library,we identified 12 unique clones with neutralization activity for SARS-CoV-2,leading to selection of monoclonal antibodies with robust neutralization activity against Delta and BA.5 variants. We applied our directed evolution approach to libraries generated by ex vivo AID-induced somatic hypermutation (SHM) of immortalized B cell clones to enhance the affinity and cross-reactivity,resulting in improved binding and neutralization potency to escape variants such as EG.5.1 and JN.1. Furthermore,we engineered a bi-paratopic antibody combining KBA2401,a broadly neutralizing antibody binding to highly conserved epitope on Spike-RBD,and KBA2402,a broadly binding non-neutralizing antibody,resulting in enhanced potency against SARS-CoV-2 variant JN.1 and KP.3.DiscussionOur findings illustrate the use of immortalized B cell libraries for development of therapeutics that adapt to viral evolution and highlight the application of ex vivo directed evolution in refining antibody responses against emerging immune escape SARS-CoV-2 variants. The approach here described offers a promising pathway for rapid therapeutic development in the face of evolving viral threats. View Publication

The selection of genetically engineered immune or hematopoietic cells in vivo after gene editing remains a clinical problem and requires a method to spare on-target toxicity to normal cells. Here,we develop a base editing approach exploiting a naturally occurring CD33 single nucleotide polymorphism leading to removal of full-length CD33 surface expression on edited cells. CD33 editing in human and nonhuman primate hematopoietic stem and progenitor cells protects myeloid progeny from CD33-targeted therapeutics without affecting normal hematopoiesis in vivo,thus demonstrating potential for improved immunotherapies with reduced off-leukemia toxicity. For broader application to gene therapies,we demonstrate highly efficient (>70%) multiplexed adenine base editing of the CD33 and gamma globin genes,resulting in long-term persistence of dual gene-edited cells with HbF reactivation in nonhuman primates. Using the CD33 antibody-drug conjugate Gemtuzumab Ozogamicin,we show resistance of engrafted,multiplex edited human cells in vivo,and a 2-fold enrichment for edited cells in vitro. Together,our results highlight the potential of adenine base editors for improved immune and gene therapies. Subject terms: Haematopoietic stem cells,Bone marrow transplantation,Cell biology View Publication

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy characterized by desmoplastic stroma,immunosuppressive tumor microenvironment (TME),and resistance to standard therapies. Natural killer (NK) cell-based immunotherapies have shown limited efficacy due to impaired persistence,infiltration,and function in PDAC. Methods: We established a direct reprogramming strategy to generate cytotoxic NK cells (1 F-NKs) by targeting BCL11B,a transcription factor essential for T cell lineage commitment,using shRNA or CRISPR/Cas9 in peripheral blood mononuclear cells (PBMCs). A genome-wide CRISPR/Cas9 screen identified tumor-intrinsic modulators of NK resistance. Functional and in vivo studies assesses the efficacy of 1 F-NKs alone and in combination with mesothelin (MSLN)-CAR engineering and PKMYT1 inhibition. Results: BCL11B depletion enabled the generation of CD56brightCD16bright 1 F-NKs with potent cytotoxicity and elevated NKG2D and CX3CR1 expression. Site-specific integration of a mesothelin (MSLN)-CAR into BCL11B locus generated MSLN-1 F-NKs with stable antigen specific activity. A genome-wide screen identified PKMYT1 as a modulator of tumor resistance to NK cell-mediated killing; its inhibition by RP6306 upregulated NKG2D ligands (MICA/B) and CX3CL1,sensitizing PDACs to 1 F-NK cytotoxicity. In PDAC xenograft models,1 F-NKs alone or combined with CAR engineering and RP6306 significantly reduced tumor growth and prolonged survival. Notably,this triple combination elicited a synergistic antitumor effect,outperforming each monotherapy or dual combination. Conclusions: This study presents a synergistic immunotherapy platform that integrates NK reprogramming,CAR engineering,and tumor sensitization. The combinatorial approach significantly enhances antitumor efficacy in PDAC and offers a promising strategy for overcoming immune resistance in solid tumors. View Publication

BACKGROUND/PURPOSE: Retinoid signaling plays an important role in many differentiation pathways. Retinoid signaling has been implicated in the induction of differentiation by pancreatic ductal cancer cell lines and in patients with pancreatic cancer. The authors wished to better understand the role of retinoid signaling in pancreatic development. METHODS: Embryonic pancreas was harvested from mice at serial gestational ages and immunohistochemical analysis was performed for retinoic acid receptors (RAR-alpha,RAR-beta,RAR-gamma),and retinoid X receptors (RXR-alpha,RXR-beta,and RXR-gamma). Also,early embryonic pancreases were cultured for 7 days with exogenous 9-cis retinoic acid (9cRA) or all-trans retinoic acid (atRA) and analyzed histologically and immunohistochemically. RESULTS: Retinoid receptors were expressed in a lineage-specific distribution,with stronger expression for many in the exocrine compartment. The receptors were not often expressed until late gestation. Exogenous 9cRA induced predominantly ducts instead of acini,plus more mature endocrine (islet) architecture. Exogenous atRA induced predominantly acini instead of ducts,with no apparent endocrine effect. CONCLUSIONS: Retinoids may have an important role in pancreatic differentiation,with a particular effect on secondary lineage selection between ductal and acinar phenotype. Because the control of ductal versus acinar differentiation has been implicated strongly in the pathogenesis of pancreatic ductal carcinoma,these results may lay the groundwork for studies in the mechanism of induced differentiation of pancreatic ductal cancer by retinoids. View Publication

Inductive signals and transcription factors involved in motor neuron generation have been identified,raising the question of whether these developmental insights can be used to direct stem cells to a motor neuron fate. We show that developmentally relevant signaling factors can induce mouse embryonic stem (ES) cells to differentiate into spinal progenitor cells,and subsequently into motor neurons,through a pathway recapitulating that used in vivo. ES cell-derived motor neurons can populate the embryonic spinal cord,extend axons,and form synapses with target muscles. Thus,inductive signals involved in normal pathways of neurogenesis can direct ES cells to form specific classes of CNS neurons. View Publication