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BackgroundThe human induced pluripotent stem cells (hiPSCs) can generate all the cells composing the human body,theoretically. Therefore,hiPSCs are thought to be a candidate source of stem cells for regenerative medicine. The major challenge of allogeneic hiPSC-derived cell products is their immunogenicity. The hypoimmunogenic cell strategy is allogenic cell therapy without using immune suppressants. Advances in gene engineering technology now permit the generation of hypoimmunogenic cells to avoid allogeneic immune rejection. In this study,we generated a hypoimmunogenic hiPSC (HyPSC) clone that had diminished expression of human leukocyte antigen (HLA) class Ia and class II and expressed immune checkpoint molecules and a safety switch.MethodsFirst,we generated HLA class Ia and class II double knockout (HLA class Ia/II DKO) hiPSCs. Then,a HyPSC clone was generated by introducing exogenous β-2-microglobulin (B2M),HLA-G,PD-L1,and PD-L2 genes,and the Rapamycin-activated Caspase 9 (RapaCasp9)-based suicide gene as a safety switch into the HLA class Ia/II DKO hiPSCs. The characteristics and immunogenicity of the HyPSCs and their derivatives were analyzed.ResultsWe found that the expression of HLA-G on the cell surface can be enhanced by introducing the exogenous HLA-G gene along with B2M gene into HLA class Ia/II DKO hiPSCs. The HyPSCs retained a normal karyotype and had the characteristics of pluripotent stem cells. Moreover,the HyPSCs could differentiate into cells of all three germ layer lineages including CD45+ hematopoietic progenitor cells (HPCs),functional endothelial cells,and hepatocytes. The HyPSCs-derived HPCs exhibited the ability to evade innate and adaptive immunity. Further,we demonstrated that RapaCasp9 could be used as a safety switch in vitro and in vivo.ConclusionThe HLA class Ia/II DKO hiPSCs armed with HLA-G,PD-L1,PD-L2,and RapaCasp9 molecules are a potential source of stem cells for allogeneic transplantation.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-024-03810-4. View Publication

AbstractBackgroundCancer‐targeted T‐cell receptor T (TCR‐T) cells hold promise in treating cancers such as hematological malignancies and breast cancers. However,approaches to obtain cancer‐reactive TCR‐T cells have been unsuccessful.MethodsHere,we developed a novel strategy to screen for cancer‐targeted TCR‐T cells using a special humanized mouse model with person‐specific immune fingerprints. Rare steady‐state circulating hematopoietic stem and progenitor cells were expanded via three‐dimensional culture of steady‐state peripheral blood mononuclear cells,and then the expanded cells were applied to establish humanized mice. The human immune system was evaluated according to the kinetics of dendritic cells,monocytes,T‐cell subsets,and cytokines. To fully stimulate the immune response and to obtain B‐cell precursor NAML‐6‐ and triple‐negative breast cancer MDA‐MB‐231‐targeted TCR‐T cells,we used the inactivated cells above to treat humanized mice twice a day every 7 days. Then,human T cells were processed for TCR β‐chain (TRB) sequencing analysis. After the repertoires had been constructed,features such as the fraction,diversity,and immune signature were investigated.ResultsThe results demonstrated an increase in diversity and clonality of T cells after treatment. The preferential usage and features of TRBV,TRBJ,and the V–J combination were also changed. The stress also induced highly clonal expansion. Tumor burden and survival analysis demonstrated that stress induction could significantly inhibit the growth of subsequently transfused live tumor cells and prolong the survival of the humanized mice.ConclusionsWe constructed a personalized humanized mouse model to screen cancer‐targeted TCR‐T pools. Our platform provides an effective source of cancer‐targeted TCR‐T cells and allows for the design of patient‐specific engineered T cells. It therefore has the potential to greatly benefit cancer treatment. Cancer‐targeted T‐cell receptor T (TCR‐T) cells hold promise in treating malignancies but with limited source. We applied steady‐state peripheral blood mononuclear cells via three‐dimensional culture to construct humanized mouse model for cancer‐targeted TCR‐T repertoire screening. View Publication

IntroductionBispecific antibodies (BsAbs) can simultaneously target two epitopes of different antigenic targets,bringing possibilities for diversity in antibody drug design and are promising tools for the treatment of cancers and other diseases. T-cell engaging bsAb is an important application of the bispecific antibody,which could promote T cell-mediated tumor cell killing by targeting tumor-associated antigen (TAA) and CD3 at the same time.MethodsThis study comprised antibodies purification,Elisa assay for antigen binding,cytotoxicity assays,T cell activation by flow cytometry in vitro and xenogenic tumor model in vivo.ResultsWe present a novel bsAb platform named PHE-Ig technique to promote cognate heavy chain (HC)-light chain (LC) pairing by replacing the CH1/CL regions of different monoclonal antibodies (mAbs) with the natural A and B chains of PHE1 fragment of Integrin β2 based on the knob-in-hole (KIH) technology. We had also verified that PHE-Ig technology can be effectively used as a platform to synthesize different desired bsAbs for T-cell immunotherapy. Especially,BCMA×CD3 PHE-Ig bsAbs exhibited robust anti-multiple myeloma (MM) activity in vitro and in vivo.DiscussionMoreover,PHE1 domain was further shortened with D14G and R41S mutations,named PHE-S,and the PHE-S-based BCMA×CD3 bsAbs also showed anti BCMA+ tumor effect in vitro and in vivo,bringing more possibilities for the development and optimization of different bsAbs. To sum up,PHE1-based IgG-like antibody platform for bsAb construction provides a novel strategy for enhanced T-cell immunotherapy. View Publication

BackgroundAllergen-specific immunotherapy (AIT) is able to restore immune tolerance to allergens in allergic patients. However,some patients do not or only poorly respond to current treatment protocols. Therefore,there is a need for deeper mechanistic insights and further improvement of treatment strategies. The relevance of the aryl hydrocarbon receptor (AhR),a ligand-dependent transcription factor,has been investigated in several inflammatory diseases,including allergic asthma. However,its potential role in AIT still needs to be addressed.MethodsA murine model of AIT in ovalbumin-induced allergic airway inflammation was performed in AhR-deficient (AhR-/-) and wild-type mice. Furthermore,AIT was combined with the application of the high-affinity AhR agonist 10-chloro-7H-benzimidazo[2,1-a]benzo[de]iso-quinolin-7-one (10-Cl-BBQ) as an adjuvant to investigate the effects of AhR activation on therapeutic outcome.ResultsAlthough AhR-/- mice suffer stronger allergic responses than wild-type mice,experimental AIT is comparably effective in both. Nevertheless,combining AIT with the administration of 10-Cl-BBQ improved therapeutic effects by an AhR-dependent mechanism,resulting in decreased cell counts in the bronchoalveolar fluid,decreased pulmonary Th2 and Th17 cell levels,and lower sIgE levels.ConclusionThis study demonstrates that the success of AIT is not dependent on the AhR. However,targeting the AhR during AIT can help to dampen inflammation and improve tolerogenic vaccination. Therefore,AhR ligands might represent promising candidates as immunomodulators to enhance the efficacy of AIT. View Publication

Cryptosporidium causes debilitating diarrheal disease in patients with primary and acquired defects in T cell function. However,it has been a challenge to understand how this infection generates T cell responses and how they mediate parasite control. Here,Cryptosporidium was engineered to express a parasite effector protein (MEDLE-2) that contains the major histocompatibility complex-I restricted SIINFEKL epitope which is recognized by T cell receptor transgenic OT-I(OVA-TCR-I) clusters of differentiation (CD)8+ T cells. These modified parasites induced expansion of endogenous SIINFEKL-specific and OT-I CD8+ T cells that were a source of interferon-gamma (IFN-γ) that could restrict growth of Cryptosporidium. This T cell response was dependent on the translocation of the effector and similar results were observed with another secreted parasite effector (rhoptry protein 1). Although infection and these translocated effector proteins are restricted to intestinal epithelial cells,type 1 conventional dendritic cells were required to generate CD8+ T cell responses to these model antigens. These data sets highlight Cryptosporidium effectors as potential targets of the immune system and suggest that crosstalk between enterocytes and type 1 conventional dendritic cells is crucial for CD8+ T cell responses to Cryptosporidium. View Publication

Siglec-6 is a lectin receptor with restricted expression in the placenta,mast cells and memory B-cells. Although Siglec-6 is expressed in patients with chronic lymphocytic leukemia (CLL),its pathophysiological role has not been elucidated. We describe here a role for Siglec-6 in migration and adhesion of CLL B cells to CLL- bone marrow stromal cells (BMSCs) in vitro and compromised migration to bone marrow and spleen in vivo. Mass spectrometry analysis revealed interaction of Siglec-6 with DOCK8,a guanine nucleotide exchange factor. Stimulation of MEC1-002 CLL cells with a Siglec-6 ligand,sTn,results in Cdc42 activation,WASP protein recruitment and F-actin polymerization,which are all associated with cell migration. Therapeutically,a Siglec-6/CD3-bispecific T-cell-recruiting antibody (T-biAb) improves overall survival in an immunocompetent mouse model and eliminates CLL cells in a patient derived xenograft model. Our findings thus reveal a migratory role for Siglec-6 in CLL,which can be therapeutically targeted using a Siglec-6 specific T-biAb. Siglec-6 is often overexpressed in chronic lymphocytic leukaemia (CLL),but its role is unclear. Here,the author report that Siglec-6 regulates the migration and adhesion of CLL B cells via interaction with sialyl Tn on bone marrow stromal cells driving invasion which could be therapeutically targeted using a Siglec-6/CD3-bispecfiic antibody. View Publication

Long interspersed element 1 (LINE-1; L1) are a family of transposons that occupy ~17% of the human genome. Though a small number of L1 copies remain capable of autonomous transposition,the overwhelming majority of copies are degenerate and immobile. Nevertheless,both mobile and immobile L1s can exert pleiotropic effects (promoting genome instability,inflammation,or cellular senescence) on their hosts,and L1’s contributions to aging and aging diseases is an area of active research. However,because of the cell type-specific nature of transposon control,the catalogue of L1 regulators remains incomplete. Here,we employ an eQTL approach leveraging transcriptomic and genomic data from the GEUVADIS and 1000Genomes projects to computationally identify new candidate regulators of L1 RNA levels in lymphoblastoid cell lines. To cement the role of candidate genes in L1 regulation,we experimentally modulate the levels of top candidates in vitro,including IL16,STARD5,HSD17B12,and RNF5,and assess changes in TE family expression by Gene Set Enrichment Analysis (GSEA). Remarkably,we observe subtle but widespread upregulation of TE family expression following IL16 and STARD5 overexpression. Moreover,a short-term 24-hour exposure to recombinant human IL16 was sufficient to transiently induce subtle,but widespread,upregulation of L1 subfamilies. Finally,we find that many L1 expression-associated genetic variants are co-associated with aging traits across genome-wide association study databases. Our results expand the catalogue of genes implicated in L1 RNA control and further suggest that L1-derived RNA contributes to aging processes. Given the ever-increasing availability of paired genomic and transcriptomic data,we anticipate this new approach to be a starting point for more comprehensive computational scans for regulators of transposon RNA levels. Author summaryTransposable elements,or jumping genes,are fragments of DNA that have or once had the ability to mobilize to a new location within our genome. In humans,the most abundant transposable element is LINE-1 (L1),accounting for ~17% of our total DNA. Though L1 is generally repressed in healthy human cells,derepression of transposable elements (including L1) has been observed in aging and in aging-associated diseases. Additionally,there is increasing evidence that L1 transcriptional levels may promote features of aging,highlighting the importance of understanding the mechanisms that regulate L1 RNA levels. Here,we computationally identify new candidate regulators of L1 RNA levels,provide experimental evidence that candidate regulators influence L1 RNA levels,and demonstrate that genetic variants associated with differences in L1 RNA levels are co-associated with aging phenotypes. Our approach expands the toolkit that can be used to characterize transposable element regulation and highlights specific genes for further study. Importantly,our results reiterate the notion that L1 levels are linked with aging phenotypes and represent a potential therapeutic target for age-related decline. View Publication

Hypoimmune gene edited human pluripotent stem cells (hPSCs) are a promising platform for developing reparative cellular therapies that evade immune rejection. Existing first-generation hypoimmune strategies have used CRISPR/Cas9 editing to modulate genes associated with adaptive (e.g.,T cell) immune responses,but have largely not addressed the innate immune cells (e.g.,monocytes,neutrophils) that mediate inflammation and rejection processes occurring early after graft transplantation. We identified the adhesion molecule ICAM-1 as a novel hypoimmune target that plays multiple critical roles in both adaptive and innate immune responses post-transplantation. In a series of studies,we found that ICAM-1 blocking or knock-out (KO) in hPSC-derived cardiovascular therapies imparted significantly diminished binding of multiple immune cell types. ICAM-1 KO resulted in diminished T cell proliferation responses in vitro and in longer in vivo retention/protection of KO grafts following immune cell encounter in NeoThy humanized mice. The ICAM-1 KO edit was also introduced into existing first-generation hypoimmune hPSCs and prevented immune cell binding,thereby enhancing the overall hypoimmune capacity of the cells. This novel hypoimmune editing strategy has the potential to improve the long-term efficacy and safety profiles of regenerative therapies for cardiovascular pathologies and a number of other diseases. Graphical Abstract ICAM-1 Knock-out in Transendothelial Migration and at the Immune Synapse. Abbreviations: PSC-EC - pluripotent stem cell-derived endothelial cells; KO – knock-out; dSMAC – distal supramolecular activation complex; pSMAC – peripheral supramolecular activation complex; cSMAC – central supramolecular activation complex. View Publication

Over 4% of the global population is estimated to live with autoimmune disease,necessitating immunosuppressive treatment that is often chronic,not curative,and carries associated risks. B cells have emerged as key players in disease pathogenesis,as evidenced by partial responsiveness to B cell depletion by antibody-based therapies. However,these treatments often have transient effects due to incomplete depletion of tissue-resident B cells. Chimeric antigen receptor (CAR) T cells targeting B cells have demonstrated efficacy in refractory systemic lupus erythematosus. To this end,we developed an anti-CD19 CAR T cell product candidate,CABA-201,containing a clinically evaluated fully human CD19 binder (IC78) with a 4-1BB costimulatory domain and CD3 zeta stimulation domain for treatment refractory autoimmune disease. Here,we demonstrate specific cytotoxic activity of CABA-201 against CD19+ Nalm6 cells with no off-target effects on primary human cells. Novel examination of CABA-201 generated from primary T cells from multiple patients with autoimmune disease displayed robust CAR surface expression and effective elimination of the intended target autologous CD19+ B cells in vitro. Together,these findings support the tolerability and activity of CABA-201 for clinical development in patients with autoimmune disease. Graphical abstract Basu and colleagues show CABA-201,a B cell-targeting CAR T cell product with a fully human CD19 binder and 4-1BB costimulatory domain,can precisely eliminate autoimmune patient B cells without off-target deleterious effects,demonstrating its ability as a robust therapeutic for B cell-driven autoimmune disorders. View Publication

Cis-regulatory elements (CREs) interact with trans regulators to orchestrate gene expression,but how transcriptional regulation is coordinated in multi-gene loci has not been experimentally defined. We sought to characterize the CREs controlling dynamic expression of the adjacent costimulatory genes CD28,CTLA4 and ICOS,encoding regulators of T cell-mediated immunity. Tiling CRISPR interference (CRISPRi) screens in primary human T cells,both conventional and regulatory subsets,uncovered gene-,cell subset- and stimulation-specific CREs. Integration with CRISPR knockout screens and assay for transposase-accessible chromatin with sequencing (ATAC-seq) profiling identified trans regulators influencing chromatin states at specific CRISPRi-responsive elements to control costimulatory gene expression. We then discovered a critical CCCTC-binding factor (CTCF) boundary that reinforces CRE interaction with CTLA4 while also preventing promiscuous activation of CD28. By systematically mapping CREs and associated trans regulators directly in primary human T cell subsets,this work overcomes longstanding experimental limitations to decode context-dependent gene regulatory programs in a complex,multi-gene locus critical to immune homeostasis. Functional characterization of the regulatory landscape of the adjacent costimulatory genes CD28,CTLA4 and ICOS in primary human T cell subsets identifies context-dependent programs controlling this locus critical for immune homeostasis. View Publication

Given the marginal penetration of most drugs across the blood-brain barrier,the efficacy of various agents remains limited for glioblastoma (GBM). Here we employ low-intensity pulsed ultrasound (LIPU) and intravenously administered microbubbles (MB) to open the blood-brain barrier and increase the concentration of liposomal doxorubicin and PD-1 blocking antibodies (aPD-1). We report results on a cohort of 4 GBM patients and preclinical models treated with this approach. LIPU/MB increases the concentration of doxorubicin by 2-fold and 3.9-fold in the human and murine brains two days after sonication,respectively. Similarly,LIPU/MB-mediated blood-brain barrier disruption leads to a 6-fold and a 2-fold increase in aPD-1 concentrations in murine brains and peritumoral brain regions from GBM patients treated with pembrolizumab,respectively. Doxorubicin and aPD-1 delivered with LIPU/MB upregulate major histocompatibility complex (MHC) class I and II in tumor cells. Increased brain concentrations of doxorubicin achieved by LIPU/MB elicit IFN-γ and MHC class I expression in microglia and macrophages. Doxorubicin and aPD-1 delivered with LIPU/MB results in the long-term survival of most glioma-bearing mice,which rely on myeloid cells and lymphocytes for their efficacy. Overall,this translational study supports the utility of LIPU/MB to potentiate the antitumoral activities of doxorubicin and aPD-1 for GBM. Ultrasound-mediated blood-brain barrier opening has been exploited to improve drug delivery in the brain. Here the authors show that low-intensity pulsed ultrasound in combination with intravenous injection of microbubbles enhances the delivery of doxorubicin and anti-PD1 in gliomas,improving anti-tumor immune responses. View Publication

Signal regulatory protein alpha (SIRPα) is an immune inhibitory receptor on myeloid cells including macrophages and dendritic cells,which binds to CD47,a ubiquitous self-associated molecule. SIRPα-CD47 interaction is exploited by cancer cells to suppress anti-tumor activity of myeloid cells,therefore emerging as a novel immune checkpoint for cancer immunotherapy. In blood cancer,several SIRPα-CD47 blockers have shown encouraging monotherapy activity. However,the anti-tumor activity of SIRPα-CD47 blockers in solid tumors seems limited,suggesting the need for combination therapies to fully exploit the myeloid immune checkpoint in solid tumors. Here we tested whether combination of SIRPα-CD47 blocker with antibody-drug conjugate bearing a topoisomerase I inhibitor DXd (DXd-ADC) would enhance anti-tumor activity in solid tumors. To this end,DS-1103a,a newly developed anti-human SIRPα antibody (Ab),was assessed for the potential combination benefit with datopotamab deruxtecan (Dato-DXd) and trastuzumab deruxtecan (T-DXd),DXd-ADCs targeting human trophoblast cell-surface antigen 2 and human epidermal growth factor receptor 2,respectively. DS-1103a inhibited SIRPα-CD47 interaction and enhanced antibody-dependent cellular phagocytosis of Dato-DXd and T-DXd against human cancer cells. In a whole cancer cell vaccination model,vaccination with DXd-treated cancer cells led to activation of tumor-specific T cells when combined with an anti-mouse SIRPα (anti-mSIRPα) Ab,implying the benefit of combining DXd-ADCs with anti-SIRPα Ab on anti-tumor immunity. Furthermore,in syngeneic mouse models,both Dato-DXd and T-DXd combination with anti-mSIRPα Ab showed stronger anti-tumor activity over the monotherapies. Taken together,this study provides a preclinical rationale of novel therapies for solid tumors combining SIRPα-CD47 blockers with DXd-ADCs. View Publication