技术资料

Chimeric antigen receptor (CAR) T cells targeting CD19 mediate potent antitumor effects in B-cell malignancies including acute lymphoblastic leukemia (ALL),but antigen loss remains the major cause of treatment failure. To mitigate antigen escape and potentially improve the durability of remission,we developed a dual-targeting approach using an optimized,bispecific CAR construct that targets both CD19 and BAFF-R. CD19/BAFF-R dual CAR T cells exhibited antigen-specific cytokine release,degranulation,and cytotoxicity against both CD19-/- and BAFF-R-/- variant human ALL cells in vitro. Immunodeficient mice engrafted with mixed CD19-/- and BAFF-R-/- variant ALL cells and treated with a single dose of CD19/BAFF-R dual CAR T cells experienced complete eradication of both CD19-/- and BAFF-R-/- ALL variants,whereas mice treated with monospecific CD19 or BAFF-R CAR T cells succumbed to outgrowths of CD19-/BAFF-R+ or CD19+/BAFF-R- tumors,respectively. Further,CD19/BAFF-R dual CAR T cells showed prolonged in vivo persistence,raising the possibility that these cells may have the potential to promote durable remissions. Together,our data support clinical translation of BAFF-R/CD19 dual CAR T cells to treat ALL. View Publication

B cell activation and Ab production in response to protein Ags requires presentation of peptides for recruitment of T cell help. We and others have recently demonstrated that B cells can also acquire innate help by presenting lipid Ags via CD1d to NKT cells. Given the newfound contribution of NKT cells to humoral immunity,we sought to identify the pathways that regulate CD1 molecule expression in human B cells. We show that ex vivo,activated and memory B cells expressed lower levels of CD1d compared with resting,naive,and marginal zone-like B cells. In vitro,CD1d was downregulated by all forms of B cell activation,leaving a narrow temporal window in which B cells could activate NKT cells. CD1c expression and function also decreased following activation by CD40L alone,whereas activation via the BCR significantly upregulated CD1c,particularly on marginal zone-like B cells. We found that the CD40L-induced downregulation of CD1d and CD1c correlated with diminished expression of retinoic acid receptor α (RARα) response genes,an effect that was reversed by RARα agonists. However,BCR-induced upregulation of CD1c was independent of the RAR pathway. Our findings that both CD1d and CD1c are upregulated by RARα signaling in human B cells is distinct from effects reported in dendritic cells,in which CD1c is inversely downregulated. One functional consequence of CD1d upregulation by retinoic acid was NKT cell cytotoxicity toward B cells. These results are central to our understanding of how CD1-restricted T cells may control humoral immunity. View Publication

Langerhans cell histiocytosis (LCH) is a rare disease with an unknown etiology characterized by heterogeneous lesions containing CD207+CD1a+ cells that can arise in almost any tissue and cause significant morbidity and mortality. Precursors of pathological Langerhans cells have yet to be defined. Our aim was to identify circulating CD207+CD1a+ cells and their inducers in LCH. Expression of CD207 and CD1a in the blood myeloid compartment as well as thymic stromal lymphopoietin (TSLP) and transforming growth factor β (TGF-β) plasma levels were measured in 22 pediatric patients with active disease (AD) or nonactive disease (NAD). In patients with AD vs those with NAD,the myeloid compartment showed an increased CD11b (CD11bhigh plus CD11b+) fraction (39.7 ± 3.6 vs 18.6 ± 1.9),a higher percentage of circulating CD11bhighCD11c+CD207+ cells (44.5 ± 11.3 vs 3.2 ± 0.5),and the presence of CD11chighCD207+CD1a+ cells (25.0 ± 9.1 vs 2.3 ± 0.5). Blood CD207+CD1a+ cells were not observed in adult controls or umbilical cord. Increased TSLP and TGF-β levels were detected in patients with AD. Interestingly,plasma from patients with AD induces CD207 expression on CD14+ monocytes. We conclude that CD207+CD1a+ cells are circulating in patients with active LCH,and TSLP and TGF-β are potential drivers of Langerhans-like cells in vivo. View Publication

Natural killer (NK) cell recognition of tumor cells is mediated through activating receptors such as CD226,with suppression of effector functions often controlled by negative regulatory transcription factors such as FOXO1. Here we show that CD226 regulation of NK cell cytotoxicity is facilitated through inactivation of FOXO1. Gene-expression analysis of NK cells isolated from syngeneic tumors grown in wild-type or CD226-deficient mice revealed dysregulated expression of FOXO1-regulated genes in the absence of CD226. In vitro cytotoxicity and stimulation assays demonstrated that CD226 is required for optimal killing of tumor target cells,with engagement of its ligand CD155 resulting in phosphorylation of FOXO1. CD226 deficiency or anti-CD226 antibody blockade impaired cytotoxicity with concomitant compromised inactivation of FOXO1. Furthermore,inhibitors of FOXO1 phosphorylation abrogated CD226-mediated signaling and effector responses. These results define a pathway by which CD226 exerts control of NK cell responses against tumors. View Publication

CD24 expression on pro-B cells plays a role in B cell selection and development in the bone marrow. We previously detected higher CD24 expression and frequency within IgD+ na{\{i}}ve and memory B cells in patients with Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) compared with age-matched healthy controls (HC). Here we investigated the relationship between CD24 expression and B cell maturation. In vitro stimulation of isolated B cells in response to conventional agonists were used to follow the dynamics of CD24 positivity during proliferation and differentiation (or maturation). The relationship between CD24 expression to cycles of proliferation and metabolism in purified B cells from HC was also investigated using phospho-flow (phosphorylation of AMPK-pAMPK) 1proton nuclear magnetic resonance and Mitotracker Far-red (Mitochondrial mass-MM). In vitro in the absence of stimulation there was an increased percentage of CD24+ viable B cells in ME/CFS patients compared to HC (p {\textless} 0.05) following 5 days culture. Following stimulation with B cell agonists percentage of CD24+B cells in both na{\"{i}}ve and memory B cell populations decreased. P {\textless} 0.01). There was a negative relationship between percentage of CD24+B cells with MM (R2 = 0.76; p {\textless} 0.01) which was subsequently lost over sequential cycles of proliferation. There was a significant correlation between CD24 expression on B cells and the usage of glucose and secretion of lactate in vitro. Short term ligation of the B cell receptor with anti-IgM antibody significantly reduced the viability of CD24+ memory B cells compared to those cross-linked by anti-IgD or anti-IgG antibody. A clear difference was found between na{\""{i}}ve and memory B cells with respect to CD24 expression and pAMPK most notably a strong positive association in IgD+IgM+ memory B cells. In vitro findings confirmed dysregulation of CD24-expressing B cells from ME/CFS patients previously suggested by immunophenotype studies of B cells from peripheral blood. CD24-negative B cells underwent productive proliferation whereas CD24+ B cells were either unresponsive or susceptible to cell death upon BCR-engagement alone. We suggest that CD24 expression may reflect variations in energy metabolism on different B cell subsets.""" View Publication

CD24 is a signal-transducing molecule on the surfaces of most human B cells that can modulate their response to activation signals by antagonizing IL-induced differentiation into antibody-forming cells and inducing proliferation in combination with signals generated by Ag receptors. A cDNA that directs the expression of CD24 on the surfaces of transfected COS cells was cloned by its homology to a cDNA encoding the murine M1/69-J11d heat stable Ag. The CD24 cDNA encodes a mature peptide of only 31 to 35 amino acids that is extensively glycosylated and is attached to the outer surface of the plasma membrane by a glycosyl phosphatidylinositol lipid anchor. Although CD24 is structurally similar to M1/69-J11d,and the two Ag appear to have a common genetic ancestry,the homology of CD24 to the M1/69-J11d Ag is confined to a small cluster of amino acids comprising potential N-linked glycosylation sites. Combined with the differences in expression patterns of the human and murine Ag,this indicates that CD24 and M1/69-J11d may not have equivalent functional roles in lymphoid development. The novel structure of CD24 suggests that signaling could be triggered by the binding of a lectin-like ligand to the carbohydrates projecting from the CD24 peptide,and transduced through the release of second messengers derived from the glycosyl phosphatidylinositol membrane anchor of CD24. View Publication

BackgroundCD24 plays a crucial role not only in promoting tumor progression and metastasis but also in modulating macrophage-mediated anti-tumor immunity. However,its impact on the immune landscape of the tumor microenvironment (TME) remains unexplored. Here,we investigated the role of CD24a,the murine CD24 gene,in tumor progression and TME immune dynamics in a murine triple-negative breast cancer (TNBC) model.MethodsClustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9 knockout technology was employed to generate CD24a knockout in the murine TNBC cell line 4T1. Flow cytometry was utilized to analyze the immune cell populations,including myeloid-derived suppressor cells (MDSCs),natural killer cells,T cells,and macrophages,within tumors,spleens,and bone marrow in the orthotopic mouse 4T1 breast cancer model. Immunofluorescence (IF) staining was used to detect the immune cells in tumor sections. High-speed confocal was used to perform three-dimensional (3D) mapping of immune cells in the 4T1 orthotopic tumors.ResultsKnocking out CD24a significantly reduced tumor growth kinetics and prolonged mouse survival in vivo. Flow cytometry and IF analysis of tumor samples revealed that CD24a loss significantly promoted the infiltration of M1 macrophages and cytotoxic CD8+ T cells into the TME while reducing the recruitment and expansion of granulocytic MDSCs (gMDSCs). In vitro coculture experiments showed that CD24a deficiency significantly enhanced macrophage‐mediated phagocytosis and CD8⁺ T cell-mediated cytotoxicity,effects that were partially reversed by re‐expression of CD24a. Moreover,in vivo depletion of macrophages and CD8+ T cells reverted the delayed tumor growth caused by CD24a knockout,underscoring their critical role in tumor growth suppression associated with CD24a knockout. 3D mapping of immune cells in the TME confirmed the anti-tumor immune landscape in the CD24a knockout 4T1 tumors. Furthermore,in vitro analysis showed that CD24a loss upregulated macrophage colony-stimulating factor expression while suppressed levels of CXCL1,CXCL5,and CXCL10,chemokines known to recruit gMDSCs,further providing a molecular basis for enhanced macrophage recruitment and diminished gMDSC accumulation.ConclusionsOur findings suggest that CD24a may regulate immune suppression within the TNBC TME. Targeting CD24a enhances macrophage- and CD8⁺ T cell-mediated anti-tumor immune responses and is associated with a shift in the TME toward a more immunogenic state,thereby suppressing tumor growth. These results may support CD24 as a promising immunotherapeutic target for TNBC.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12929-025-01165-3. View Publication

Activation of natural killer (NK) cell cytotoxicity requires adhesion and formation of a conjugate with a susceptible target cell,followed by actin polymerization,and polarization of the microtubule organizing center (MTOC) and cytolytic granules to the NK cell immune synapse. Here,by using the YTS NK cell line as a model,CD28 is shown to be an activating receptor. It signals cytotoxicity in a process dependent on phosphoinositide-3 kinase activation,leading to sustained extracellular signal-regulated kinase 2 (ERK2) phosphorylation. ERK and phospho-ERK localize to microtubule filaments. Neither conjugation with targets nor actin polymerization is affected by blocking ERK2 activation. However,both polarization of the MTOC and cytolytic granules to the synaptic region and NK cell cytotoxicity are strongly reduced by blocking ERK2 activation. A role for the CD28/CD80 interaction in cytotoxicity of human peripheral NK cells also was established. By contrast,lymphocyte function-associated antigen 1 (LFA-1) ligation transduces only a transient ERK2 activation and fails to induce killing in YTS cells. Thus,in YTS cells,a CD28 signal is used to polarize the MTOC and cytolytic granules to the NK cell immune synapse by stimulating sustained ERK2 activation. View Publication

Naïve T cells require B7/CD28 costimulation in order to be fully activated. Attempts to block this pathway have been effective in preventing unwanted immune reactions. As B7 blockade might also affect Treg cells and interfere with negative signaling through membrane CTLA-4 on effector T (Teff) cells,its immune-modulatory effects are potentially more complex. Here,we used the mouse model of multiple sclerosis (MS),EAE,to study the effect of B7 blockade. An effective therapy for MS patients has to interfere with ongoing inflammation,and therefore we injected CTLA-4Ig at day 7 and 9 after immunization,when myelin-reactive T cells have been primed and start migrating toward the CNS. Surprisingly,B7 blockade exacerbated disease signs and resulted in more severe CNS inflammation and demyelination,and was associated with an enhanced production of the inflammatory cytokines IL-17 and IFN-γ. Importantly,CTLA-4Ig treatment resulted in a transient reduction of Ki67 and CTLA-4 expression and function of peripheral Treg cells. Taken together,B7 blockade at a particular stage of the autoimmune response can result in the suppression of Treg cells,leading to a more severe disease. View Publication

Cultures of induced pluripotent stem cells (iPSCs) often contain cells of varying grades of pluripotency. We present novel lentiviral vectors targeted to the surface receptor CD30 (CD30-LV) to transfer genes into iPSCs that are truly pluripotent as demonstrated by marker gene expression. We demonstrate that CD30 expression is restricted to SSEA4high cells of human iPSC cultures and a human embryonic stem cell line. When CD30-LV was added to iPSCs during routine cultivation,efficient and exclusive transduction of cells positive for the pluripotency marker Oct-4 was achieved,while retaining their pluripotency. When added during the reprogramming process,CD30-LV solely transduced cells that became fully reprogrammed iPSCs as confirmed by co-expression of endogenous Nanog and the reporter gene. Thus,CD30-LV may serve as novel tool for the selective gene transfer into pluripotent stem cells with broad applications in basic and therapeutic research. View Publication

Although CD31 expression on human thymocytes has been reported,a detailed analysis of CD31 expression at various stages of T cell development in the human thymus is missing. In this study,we provide a global picture of the evolution of CD31 expression from the CD34(+) hematopoietic precursor to the CD45RA(+) mature CD4(+) and CD8(+) single-positive (SP) T cells. Using nine-color flow cytometry,we show that CD31 is highly expressed on CD34(+) progenitors and stays high until the early double-positive stage (CD3(-)CD4(+)CD8α(+)β(-)). After β-selection,CD31 expression levels become low to undetectable. CD31 expression then increases and peaks on CD3(high)CD4(+)CD8(+) double-positive thymocytes. However,following positive selection,CD31 expression differs dramatically between CD4(+) and CD8(+) lineages: homogeneously high on CD8 SP but lower or negative on CD4 SP cells,including a subset of CD45RA(+)CD31(-) mature CD4(+) thymocytes. CD31 expression on TCRγδ thymocytes is very similar to that of CD4 SP cells. Remarkably,there is a substantial subset of semimature (CD45RA(-)) CD4 SP thymocytes that lack CD31 expression. Moreover,FOXP3(+) and ICOS(+) cells are overrepresented in this CD31(-) subpopulation. Despite this CD31(-)CD45RA(-) subpopulation,most egress-capable mature CD45RA(+) CD4 SP thymocytes express CD31. The variations in CD31 expression appear to coincide with three major selection processes occurring during thymopoiesis: β-selection,positive selection,and negative selection. Considering the ability of CD31 to modulate the TCR's activation threshold via the recruitment of tyrosine phosphatases,our results suggest a significant role for CD31 during T cell development. View Publication

BackgroundAlthough both pre-clinical and clinical studies show promising outcomes,resulting in rapid growth of clinical trials of MSC-based therapies in recent years,the heterogeneity and therapeutic inconsistency of MSCs have severely hampered their clinical applications. Purifying homogenous MSC populations with enhanced specific functions represents one promising approach. We have demonstrated recently that the CD317+ MSCs have enhanced anti-inflammatory functions and improved therapeutic efficacy and consistency.MethodsIn the current study,we performed both in vitro and in vivo investigations to delineate whether and how CD317 regulates the immune modulation function of MSCs.ResultsOur data here indicate that the CD317 directly contributes to the immune suppression function of MSCs stimulated by TNF-α through up-regulating TSG6 via CD317/lipid-raft/TNFR1 complex. The CD317 stabilizes the TNFR1 complex,resulting in hyper-activation of the NF-κB pathway and up-regulation of TSG6,which confers the therapeutic effects of MSCs on the mouse model of ALI (acute lung injury) and IBD (inflammatory bowel disease).ConclusionsThus,the CD317 stabilizes TNFR1 and confers the anti-inflammatory functions of MSCs via NF-κB/TSG6 Pathway.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-025-04527-8. View Publication