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BackgroundAnti-CD19 chimeric antigen receptor T (CAR-T) cell therapy has proven effective for treating relapsed or refractory acute B cell leukemia. However,challenges such as cytokine release syndrome,T cell dysfunction,and exhaustion persist. Enhancing CAR-T cell efficacy through changing CAR internalization and recycling is a promising approach. The transmembrane domain is the easiest motif to optimize for modulating CAR internalization and recycling without introducing additional domains,and its impact on CAR internalization and recycling has not yet been thoroughly explored. In this study,we aim to enhance CAR-T cell function by focusing on the solely transmembrane domain design.MethodsUtilizing plasmid construction and lentivirus generation,we get two different transmembrane CAR-T cells [19CAR-T(1a) and 19CAR-T(8α)]. Through co-culture with tumor cells,we evaluate CAR dynamic change,activation levels,exhaustion markers,mitochondrial function,and differentiation in both CAR-T cells. Furthermore,immunofluorescence microscopy analysis is performed to reveal the localization of internalized CAR molecules. RNA sequencing is used to detect the transcriptome of activated CAR-T cells. Finally,a mouse study is utilized to verify the anti-tumor efficacy of 19CAR-T(1a) cells in vivo.ResultsOur findings demonstrate that 19CAR-T(1a) has lower surface CAR expression,faster internalization,and a higher recycling rate compared to 19CAR-T(8α). Internalized 19CAR(1a) co-localizes more with early and recycling endosomes,and less with lysosomes than 19CAR(8α). These features result in lower activation levels,less cytokine release,and reduced exhaustion markers in 19CAR-T(1a). Furthermore,CAR-T cells with CD1a transmembrane domain also exhibit a superior anti-tumor ability and reduced exhaustion in vivo.ConclusionOverall,we demonstrate that the transmembrane domain plays a critical role in CAR-T cell function. An optimized transmembrane domain can alleviate cytokine release syndrome and reduce CAR-T cell exhaustion,providing a direction for CAR design to enhance CAR-T cell function. View Publication

CD4+ T cells have been well-regarded as “helper” cells in activating the cytotoxicity of CD8+ T cells for effective tumor eradication,while few studies have focused on whether CD8+ T cells regulate CD4+ T cells. Our previous studies provided evidence for an interaction between CD4+ and CD8+ T cells after cryo-thermal therapy,but the mechanism remains unclear,especially pertaining to how CD8+ T cells promote the Th1 differentiation of CD4+ T cells. This study revealed that activated CD4+ and CD8+ T cells are critical for CTT-induced antitumor immunity,and the interaction between activated T cells is enhanced. The reciprocal regulation of activated CD8+ and CD4+ T cells was through LFA-1/ICAM-1 interactions,in which CD8+ T cells facilitate Notch1-dependent CD4+ Th1-dominant differentiation and promote IL-2 secretion of CD4+ T cells. Meanwhile,IL-2 derived from CD4+ T cells enhances the cytotoxicity of CD8+ T cells and establishes a positive feedback loop via increasing the expression of LFA-1 and ICAM-1 on T cells. Clinical analyses further validated that LFA-1/ICAM interactions between CD4+ and CD8+ T cells are correlated with clinical outcomes. Our study extends the functions of the LFA-1/ICAM-1 adhesion pathway,indicating its novel role in the interaction of CD4+ and CD8+ T cells. View Publication

ObjectiveThis study investigated the impact of innate lymphoid cell type 2 (ILC2s) on the function of regulatory T cells (Treg) and CD8+ T cells in chronic lymphocytic leukemia (CLL) through IL-9.MethodsPeripheral blood samples were collected from CLL patients (n = 52) and healthy controls (n = 30). ILC2 proportions and IL-9 levels were assessed using flow cytometry and ELISA. Immunofluorescence staining was performed to stain GATA3,CRTH2,and IL-9 in cervical lymph nodes from CLL patients (n = 10) and control subjects with reactive lymphadenitis (n = 10). Correlation analysis between ILC2s and IL-9 was conducted using the Spearman test. ILC2s were sorted and cultured from CLL patients,followed by co-culture experiments with PBMCs of healthy controls and MEC-1 cells,with or without anti-IL-9 antibody intervention. Flow cytometry was used to measure the proportions of ILC2s,Treg cells,PD-1+/TIGIT+/CTLA-4+ Treg subsets,and granzyme B+/perforin+ CD8+ T cells,along with MEC-1 cell apoptosis.ResultsThe proportions of ILC2s and Treg,along with serum IL-9 levels,were significantly elevated in CLL patients (P < 0.05). Peripheral blood ILC2s were positively correlated with IL-9 (r = 0.609,P < 0.001). The average fluorescence intensity of GATA3,CRTH2,and IL-9 in the cervical lymph nodes of CLL patients increased significantly (P < 0.001),and IL-9 showed colocalization with GATA3 and CRTH2. In vitro,IL-9 levels in the supernatant of sorted ILC2s from CLL patients increased. Treatment with anti-IL-9 antibody significantly reduced the PD-1+ Treg and TIGIT+ Treg cells while increasing granzyme B+ CD8+ T cells (P < 0.05). However,there was no significant effect on Treg,CTLA-4+ Treg,and perforin+ CD8+ T cells (P > 0.05). Additionally,anti-IL-9 antibody significantly increased early apoptosis (P < 0.05).ConclusionILC2s affect CD8+ T cells and Treg cells through IL-9,weakening the anti-tumor effects of CD8+ T cells and enhancing the immunosuppressive effects of Treg cells,thereby contributing to CLL pathogenesis.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00262-025-04082-4. View Publication

Memory T cells are a highly heterogeneous collection of antigen-experienced cells that undergo dynamic adaptations upon antigen re-encounter and environmental signals. This heterogeneity hinders studies on memory T cell durability and age-related dysfunction. Using chronic Epstein-Barr virus (EBV) infection and barcode-enabled antigen tracing,we assess the influence of age on memory states at the level of single antigen-specific CD8+ T cells. In young adults (<40 years),EBV-specific CD8+ T cells recognizing different antigenic peptides assume divergent preferred differentiation phenotypes. In older adults (>65-years),antigen-specific cells show largely distinct phenotypic and transcriptomic aging trajectories. Common to many albeit not all antigen-specific populations are maintained TCR diversity,gained natural killer cell-like,innate signatures and lost stem-like features while no evidence is seen for cellular senescence or exhaustion. TCR avidity contributes to these phenotypic differences and aging-related changes. Collectively,our data uncover divergent antigen-guided aging shifts in memory T cell phenotypes,which are informative for antigen selection in optimizing vaccine design and adoptive T cell therapy. Homeostasis of memory T cells is modulated by each antigen encounter,thereby creating a heterogeneous population preventing precise tracking. Here,the authors use barcode-assisted tracing of Epstein-Barr virus-specific CD8+ memory T cells of young and older individuals to find antigen-guided,clonally divergent aging trajectories. View Publication

SummaryHIV-1 latency results from tightly regulated molecular processes that act at distinct steps of HIV-1 gene expression. Here,we characterize PCI domain-containing 2 (PCID2) protein,a subunit of the transcription and export complex 2 (TREX2) complex,to enforce transcriptional repression and post-transcriptional blocks to HIV-1 gene expression during latency. PCID2 bound the latent HIV-1 LTR (long terminal repeat) and repressed transcription initiation during latency. Depletion of PCID2 remodeled the chromatin landscape at the HIV-1 promoter and resulted in transcriptional activation and latency reversal. Immunoprecipitation coupled to mass spectrometry identified PCID2-interacting proteins to include negative viral RNA (vRNA) splicing regulators,and PCID2 depletion resulted in over-splicing of intron-containing vRNA in cell lines and primary cells obtained from PWH. MCM3AP and DSS1,two other RNA-binding TREX2 complex subunits,also inhibit transcription initiation and vRNA alternative splicing during latency. Thus,PCID2 is a novel HIV-1 latency-promoting factor,which in context of the TREX2 sub-complex PCID2-DSS1-MCM3AP blocks transcription and dysregulates vRNA processing. Graphical abstract Highlights•PCID2 is bound to the latent HIV-1 LTR as a transcriptional repressor•PCID2 enforces latency by acting on transcription initiation•PCID2 establishes blocks to alternative splicing during HIV-1 latency•PCID2 misregulates alternative splicing in cells obtained from people with HIV-1 Virology; Cell biology View Publication

BackgroundSystemic sclerosis (SSc) is an autoimmune connective tissue disease characterized by vasculopathy and progressive fibrosis of skin and several internal organs,including lungs. Macrophages are the main cells involved in the immune-inflammatory damage of skin and lungs,and alternatively activated (M2) macrophages seem to have a profibrotic role through the release of profibrotic cytokines (IL10) and growth factors (TGFβ1). Nintedanib is a tyrosine kinase inhibitor targeting several fibrotic mediators and it is approved for the treatment of SSc-related interstitial lung disease (ILD). The study aimed to evaluate the effect of nintedanib in downregulating the profibrotic M2 phenotype in cultured monocyte-derived macrophages (MDMs) obtained from SSc-ILD patients.MethodsFourteen SSc patients,fulfilling the 2013 ACR/EULAR criteria for SSc,10 SSc patients affected by ILD (SSc-ILD pts),4 SSc patients non affected by ILD (SSc pts no-ILD),and 5 voluntary healthy subjects (HSs),were recruited at the Division of Clinical Rheumatology-University of Genova,after obtaining Ethical Committee approval and patients’ informed consent. Monocytes were isolated from peripheral blood,differentiated into MDMs,and then maintained in growth medium without any treatment (untreated cells),or treated with nintedanib (0.1 and 1µM) for 3,16,and 24 h. Gene expression of macrophage scavenger receptors (CD204,CD163),mannose receptor-1 (CD206),Mer tyrosine kinase (MerTK),identifying M2 macrophages,together with TGFβ1 and IL10,were evaluated by quantitative real-time polymerase chain reaction. Protein synthesis was investigated by Western blotting and the level of active TGFβ1 was evaluated by ELISA. Statistical analysis was carried out using non-parametric Wilcoxon test.ResultsCultured untreated SSc-ILD MDMs showed a significant increased protein synthesis of CD206 (p < 0.05),CD204,and MerTK (p < 0.01),together with a significant upregulation of the gene expression of MerTK and TGFβ1 (p < 0.05; p < 0.01) compared to HS-MDMs. Moreover,the protein synthesis of CD206 and MerTK and the gene expression of TGFβ1 were significantly higher in cultured untreated MDMs from SSc-ILD pts compared to MDMs without ILD (p < 0.05; p < 0.01). In cultured SSc-ILD MDMs,nintedanib 0.1 and 1µM significantly downregulated the gene expression and protein synthesis of CD204,CD206,CD163 (p < 0.05),and MerTK (p < 0.01) compared to untreated cells after 24 h of treatment. Limited to MerTK and IL10,both nintedanib concentrations significantly downregulated their gene expression already after 16 h of treatment (p < 0.05). In cultured SSc-ILD MDMs,nintedanib 0.1 and 1µM significantly reduced the release of active TGFβ1 after 24 h of treatment (p < 0.05 vs. untreated cells).ConclusionsIn cultured MDMs from SSc-ILD pts,nintedanib seems to downregulate the profibrotic M2 phenotype through the significant reduction of gene expression and protein synthesis of M2 cell surface markers,together with the significant reduction of TGFβ1 release,and notably MerTK,a tyrosine kinase receptor involved in lung fibrosis.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13075-024-03308-7. View Publication

The corneal epithelium acts as a barrier to pathogens entering the eye; corneal epithelial cells are continuously renewed by uni-potent,quiescent limbal stem cells (LSCs) located at the limbus,where the cornea transitions to conjunctiva. There has yet to be a consensus on LSC markers and their transcriptome profile is not fully understood,which may be due to using cadaveric tissue without an intact stem cell niche for transcriptomics. In this study,we addressed this problem by using single nuclei RNA sequencing (snRNAseq) on healthy human limbal tissue that was immediately snap-frozen after excision from patients undergoing cataract surgery. We identified the quiescent LSCs as a sub-population of corneal epithelial cells with a low level of total transcript counts. Moreover,TP63,KRT15,CXCL14,and ITGβ4 were found to be highly expressed in LSCs and transiently amplifying cells (TACs),which constitute the corneal epithelial progenitor populations at the limbus. The surface markers SLC6A6 and ITGβ4 could be used to enrich human corneal epithelial cell progenitors,which were also found to specifically express the putative limbal progenitor cell markers MMP10 and AC093496.1. View Publication

Allogeneic CAR T–cell therapies are being developed for hematologic malignancies. The authors implement a Cas12a chRDNA platform to generate allogeneic immune-cloaked BCMA-specific CAR T cells with resistance to host response–mediated rejection for evaluation in multiple myeloma. AbstractAllogeneic chimeric antigen receptor (CAR) T cell therapies hold the potential to overcome many of the challenges associated with patient-derived (autologous) CAR T cells. Key considerations in the development of allogeneic CAR T cell therapies include prevention of graft-vs-host disease (GvHD) and suppression of allograft rejection. Here,we describe preclinical data supporting the ongoing first-in-human clinical study,the CaMMouflage trial (NCT05722418),evaluating CB-011 in patients with relapsed/refractory multiple myeloma. CB-011 is a hypoimmunogenic,allogeneic anti–B-cell maturation antigen (BCMA) CAR T cell therapy candidate. CB-011 cells feature 4 genomic alterations and were engineered from healthy donor–derived T cells using a Cas12a CRISPR hybrid RNA–DNA (chRDNA) genome-editing technology platform. To address allograft rejection,CAR T cells were engineered to prevent endogenous HLA class I complex expression and overexpress a single-chain polyprotein complex composed of beta-2 microglobulin (B2M) tethered to HLA-E. In addition,T-cell receptor (TCR) expression was disrupted at the TCR alpha constant locus in combination with the site-specific insertion of a humanized BCMA-specific CAR. CB-011 cells exhibited robust plasmablast cytotoxicity in vitro in a mixed lymphocyte reaction in cell cocultures derived from patients with multiple myeloma. In addition,CB-011 cells demonstrated suppressed recognition by and cytotoxicity from HLA-mismatched T cells. CB-011 cells were protected from natural killer cell–mediated cytotoxicity in vitro and in vivo due to endogenous promoter-driven expression of B2M–HLA-E. Potent antitumor efficacy,when combined with an immune-cloaking armoring strategy to dampen allograft rejection,offers optimized therapeutic potential in multiple myeloma. See related Spotlight by Caimi and Melenhorst,p. 385 View Publication

Midnolin is an essential gene with previously unknown effects in vivo. This paper shows that midnolin stimulates proteasome activity necessary for lymphopoiesis and B cell cancer growth in mice. In a genetic screen,we identified two viable missense alleles of the essential gene Midnolin (Midn) that were associated with reductions in peripheral B cells. Causation was confirmed in mice with targeted deletion of four of six MIDN protein isoforms. MIDN was expressed predominantly in lymphocytes where it augmented proteasome activity. We showed that purified MIDN directly stimulated 26S proteasome activity in vitro in a manner dependent on the ubiquitin-like domain and a C-terminal region. MIDN-deficient B cells displayed aberrant activation of the IRE-1/XBP-1 pathway of the unfolded protein response. Partial or complete MIDN deficiency strongly suppressed Eμ-Myc–driven B cell leukemia and the antiapoptotic effects of Eμ-BCL2 on B cells in vivo and induced death of Sp2/0 hybridoma cells in vitro,but only partially impaired normal lymphocyte development. Thus,MIDN is required for proteasome activity in support of normal lymphopoiesis and is essential for malignant B cell proliferation over a broad range of differentiation states. View Publication

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by anti-nuclear autoantibodies whose production is promoted by autoreactive T follicular helper (TFH) cells. During SLE pathogenesis,basophils accumulate in secondary lymphoid organs (SLO),amplify autoantibody production and disease progression through mechanisms that remain to be defined. Here,we provide evidence for a direct functional relationship between TFH cells and basophils during lupus pathogenesis,both in humans and mice. PD-L1 upregulation on basophils and IL-4 production are associated with TFH and TFH2 cell expansions and with disease activity. Pathogenic TFH cell accumulation,maintenance,and function in SLO were dependent on PD-L1 and IL-4 in basophils,which induced a transcriptional program allowing TFH2 cell differentiation and function. Our study establishes a direct mechanistic link between basophils and TFH cells in SLE that promotes autoantibody production and lupus nephritis. Basophils have been implicated in systemic lupus erythematosus (SLE),as evidenced by the fact that basophil-deficient mice do not develop the disease. Here,the authors demonstrate that PD-L1 and IL-4 expression in basophils promotes the pathogenic accumulation of follicular helper T cells in patients with SLE and murine models. View Publication

Manufacturing chimeric antigen receptor (CAR) T cell therapies is complex,with limited understanding of how medium composition impacts T cell phenotypes. CRISPR-Cas9 ribonucleoproteins can precisely insert a CAR sequence while disrupting the endogenous T cell receptor alpha constant (TRAC) gene resulting in TRAC-CAR T cells with an enriched stem cell memory T cell population,a process that could be further optimized through modifications to the medium composition. In this study we generated anti-GD2 TRAC-CAR T cells using "metabolic priming" (MP),where the cells were activated in glucose/glutamine-low medium and then expanded in glucose/glutamine-high medium. T cell products were evaluated using spectral flow cytometry,metabolic assays,cytokine production,cytotoxicity assays in vitro,and potency against human GD2+ xenograft neuroblastoma models in vivo. Compared with standard TRAC-CAR T cells,MP TRAC-CAR T cells showed less glycolysis,higher CCR7/CD62L expression,more bound NAD(P)H activity,and reduced IFN-γ,IL-2,IP-10,IL-1β,IL-17,and TGF-β production at the end of manufacturing ex vivo,with increased central memory CAR T cells and better persistence observed in vivo. MP with medium during CAR T cell biomanufacturing can minimize glycolysis and enrich memory phenotypes ex vivo,which could lead to better responses against solid tumors in vivo. Graphical abstract Cappabianca and colleagues manufactured virus-free CAR T cells at scale with CRISPR-Cas9 and “metabolically primed” them by attenuating activation in low-glucose/glutamine medium with expansion in high-glucose/glutamine medium. Priming made CAR T cells with increased stem cell memory properties,including enriched central memory phenotypes in vivo while lysing solid tumors. View Publication

Only a minority of cancer patients benefit from immune checkpoint blockade therapy. Sophisticated cross-talk among different immune checkpoint pathways as well as interaction pattern of immune checkpoint molecules carried on circulating small extracellular vesicles (sEV) might contribute to the low response rate. Here we demonstrate that PD-1 and CD80 carried on immunocyte-derived sEVs (I-sEV) induce an adaptive redistribution of PD-L1 in tumour cells. The resulting decreased cell membrane PD-L1 expression and increased sEV PD-L1 secretion into the circulation contribute to systemic immunosuppression. PD-1/CD80+ I-sEVs also induce downregulation of adhesion- and antigen presentation-related molecules on tumour cells and impaired immune cell infiltration,thereby converting tumours to an immunologically cold phenotype. Moreover,synchronous analysis of multiple checkpoint molecules,including PD-1,CD80 and PD-L1,on circulating sEVs distinguishes clinical responders from those patients who poorly respond to anti-PD-1 treatment. Altogether,our study shows that sEVs carry multiple inhibitory immune checkpoints proteins,which form a potentially targetable adaptive loop to suppress antitumour immunity. Immune checkpoint inhibition is a successful form of immune therapy; however response rates vary widely among individual patients. Here authors show that circulating small extracellular vesicles might contribute to poor response to anti-PD-1 treatment by carrying PD-1 and CD80 which results in higher level of vesicular PD-L1 expression in the circulation at the expense of expression on tumour cell membranes,causing immunosuppression. View Publication