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The limited ability of the heart to regenerate has prompted development of new systems to produce cardiomyocytes for therapeutics. While differentiation of human embryonic stem cells (hESCs) into cardiomyocytes has been well documented,the process remains inefficient and/or expensive,and progress would be facilitated by better understanding the early genetic events that cause cardiac specification. By maintaining a transgenic cardiac-specific MYH6-monomeric red fluorescent protein (mRFP) reporter hESC line in conditions that promote pluripotency,we tested the ability of combinations of 15 genes to induce cardiac specification. Screening identified GATA4 plus TBX5 as the minimum requirement to activate the cardiac gene regulatory network and produce mRFP(+) cells,while a combination of GATA4,TBX5,NKX2.5,and BAF60c (GTNB) was necessary to generate beating cardiomyocytes positive for cTnI and α-actinin. Including the chemotherapeutic agent,Ara-C,from day 10 of induced differentiation enriched for cTnI/α-actinin double positive cells to 45%. Transient expression of GTNB for 5-7 days was necessary to activate the cardiogenesis through progenitor intermediates in a manner consistent with normal heart development. This system provides a route to test the effect of different factors on human cardiac differentiation and will be useful in understanding the network failures that underlie disease phenotypes. View Publication

Differentiation of pluripotent and lineage restricted stem cells such as neural stem cells (NSCs) was studied on conducting substrates of various nature without perturbation of the genome with exogenous genetic material or chemical stimuli. Primary mouse adult neural stem cells (NSCs) and P19 pluripotent embryonal (P19 EC) carcinoma cells were used. Expression levels of neuronal markers β-III-tubulin and neurofilament were evaluated by immunochemistry and flow cytometry. It was shown that the ability of the substrate to induce differentiation directly correlated with its conductivity. Conducting substrates (conducting oxides or doped pi-conjugated organic polymers) with different morphology,structure,and conductivity mechanisms all promoted differentiation of NSC and P19 cells into neuronal lineage to a similar degree without use of additional factors such as poly-L-ornithine coating or retinoic acid,as verified by their morphology and upregulation of the neuronal markers but not astrocyte marker GFAP. However,substrates with low conductance below ca. 10(-4) S cm(-2) did not show this ability. Morphology of differentiating cells was visualized by atomic force microscopy. NSCs cells increased β-III-tubulin expression by 95% and P19 cells by over 30%. Our results suggest that the substrate conductivity is a key factor governing the cell fate. Differentiation of P19 cells into neuronal lineage on conducting substrates was attributed to downregualtion of Akt signaling pathway and increase in expression of dual oxidase 1 (DUOX 1). View Publication

Systemic sclerosis (SSc) is a life-threatening chronic connective tissue disease with the characteristics of skin fibrosis,vascular injury,and inflammatory infiltrations. Though inhibition of phosphodiesterase 4 (PDE4) has been turned out to be an effective strategy in suppressing inflammation through promoting the accumulation of intracellular cyclic adenosine monophosphate (cAMP),little is known about the functional modes of inhibiting PDE4 by apremilast on the process of SSc. The present research aimed to investigate the therapeutic effects and underlying mechanism of apremilast on SSc. Herein,we found that apremilast could markedly ameliorate the pathological manifestations of SSc,including skin dermal thickness,deposition of collagens,and increased expression of $\alpha$-SMA. Further study demonstrated that apremilast suppressed the recruitment and activation of macrophages and T cells,along with the secretion of inflammatory cytokines,which accounted for the effects of apremilast on modulating the pro-fibrotic processes. Interestingly,apremilast could dose-dependently inhibit the activation of M1 and T cells in vitro through promoting the phosphorylation of CREB. In summary,our research suggested that inhibiting PDE4 by apremilast might provide a novel therapeutic option for clinical treatment of SSc patients. View Publication

The clinical application of T cells engineered with chimeric antigen receptors (CARs) for solid tumors is challenging. A major reason for this involves tumor immune evasion mechanisms,including the high expression of immune checkpoint molecules,such as the programmed death 1 (PD-1) ligands PD-L1 and PD-L2. The inducible expression of PD-L1 in tumors has been observed after CAR-T-cell infusion,even in tumors natively not expressing PD-L1. Furthermore,numerous types of pediatric cancer do not have suitable targets for CAR-T-cell therapy. Therefore,the present study aimed to develop novel CAR-T cells that target PD-L1 and PD-L2,and to evaluate their efficacy against pediatric solid tumors. A novel CAR harboring the immunoglobulin V-set domain of the human PD-1 receptor as an antigen binding site (PD-1 CAR-T) was developed without using a single-chain variable fragment. PD-1 CAR-T cells were successfully manufactured by adding an anti-PD-1 antibody,nivolumab,to the ex vivo expansion culture to prevent fratricide during the manufacturing process due to the inducible expression of PD-L1 in activated human T cells. The expression of PD-L1 (and PD-L2 to a lesser extent) was revealed to be highly upregulated in various pediatric solid tumor cells,which displayed no or very low expression initially,on in vitro exposure to interferon-γ and/or tumor necrosis factor-α,which are cytokines secreted by tumor-infiltrating T cells. Furthermore,PD-1 CAR-T cells exhibited strong cytotoxic activity against pediatric solid tumor cells expressing PD-L1 and PD-L2. Conversely,the effect of PD-1 CAR-T cells was significantly attenuated against PD-L1-positive cells coexpressing CD80,suggesting that the toxicity of PD-1 CAR-T cells to normal immune cells,including antigen presenting cells,can be minimized. In conclusion,PD-1 ligands are promising therapeutic targets for pediatric solid tumors. PD-1 CAR-T cells,either alone or in combination with CAR-T cells with other targets,represent a potential treatment option for solid tumors. View Publication

T cell-derived cancers are hallmarked by heterogeneity,aggressiveness,and poor clinical outcomes. Available targeted therapies are severely limited due to a lack of target antigens that allow discrimination of malignant from healthy T cells. Here,we report a novel approach for the treatment of T cell diseases based on targeting the clonally rearranged T cell receptor displayed by the cancerous T cell population. As a proof of concept,we identified an antibody with unique specificity toward a distinct T cell receptor (TCR) and developed antibody-drug conjugates,precisely recognizing and eliminating target T cells while preserving overall T cell repertoire integrity and cellular immunity. Our anti-TCR antibody-drug conjugates demonstrated effective receptor-mediated cell internalization,associated with induction of cancer cell death with strong signs of apoptosis. Furthermore,cell proliferation-inhibiting bystander effects observed on target-negative cells may contribute to the molecules’ anti-tumor properties precluding potential tumor escape mechanisms. To our knowledge,this represents the first anti-TCR antibody-drug conjugate designed as custom-tailored immunotherapy for T cell-driven pathologies. Graphical abstract Harald Kolmar and colleagues report a novel approach for the treatment of the difficult-to-treat T cell lymphoma/leukemia based on targeting the clonally rearranged T cell receptor expressed by the malignant T cell population. The developed antibody-drug conjugates precisely eliminate target T cells while preserving the integrity of the T cell repertoire and cellular immunity. View Publication

Prostate cancer (PCa) ranks as the second leading cause of cancer-related deaths among men in the United States. Prostate-specific membrane antigen (PSMA) represents a well-established biomarker of PCa,and its levels correlate positively with the disease progression,culminating at the stage of metastatic castration-resistant prostate cancer. Due to its tissue-specific expression and cell surface localization,PSMA shows superior potential for precise imaging and therapy of PCa. Antibody-based immunotherapy targeting PSMA offers the promise of selectively engaging the host immune system with minimal off-target effects. Here we report on the design,expression,purification,and characterization of a bispecific engager,termed 5D3-CP33,that efficiently recruits macrophages to the vicinity of PSMA-positive cancer cells mediating PCa death. The engager was engineered by fusing the anti-PSMA 5D3 antibody fragment to a cyclic peptide 33 (CP33),selectively binding the Fc gamma receptor I (FcγRI/CD64) on the surface of phagocytes. Functional parts of the 5D3-CP33 engager revealed a nanomolar affinity for PSMA and FcγRI/CD64 with dissociation constants of KD =  3 nM and KD =  140 nM,respectively. At a concentration as low as 0.3 nM,the engager was found to trigger the production of reactive oxygen species by U937 monocytic cells in the presence of PSMA-positive cells. Moreover,flow cytometry analysis demonstrated antibody-dependent cell-mediated phagocytosis of PSMA-positive cancer cells by U937 monocytes when exposed to 0.15 nM 5D3-CP33. Our findings illustrate that 5D3-CP33 effectively and specifically activates monocytes upon PSMA-positive target engagement,resulting in the elimination of tumor cells. The 5D3-CP33 engager can thus serve as a promising lead for developing new immunotherapy tools for the efficient treatment of PCa. View Publication

Human respiratory syncytial virus (RSV) is a major cause of serious respiratory tract infections in infants and the elderly. Recently it was shown that the RSV G glycoprotein mediates attachment to cells using CX3CR1 as a receptor,and that G-specific neutralizing antibodies can be detected using human airway epithelial (HAE) cell cultures. To investigate the contributions of G-specific antibodies to RSV neutralization,we performed HAE neutralization assays on sera from RSV G-immunized mice or RSV-infected infants. We confirmed that G-specific neutralization using serum from mice or humans could only be detected on HAE cultures. We also found that RSV G-specific antibodies in infants were either subgroup specific or cross-neutralizing. Altogether,our results suggest that G is an important target for generating neutralizing antibodies and would be beneficial to include in an RSV vaccine. Further,inclusion of G antigens from both RSV subgroups may enhance the vaccine cross protection potency. View Publication

Adoptive cell therapy (ACT) targeting tumor-specific antigens holds promise for solid tumors,but limited neoantigen presentation remains a key barrier to efficacy. Here,we identify and characterize a T cell receptor (TCR),T104,for the KRAS.G12V mutation,a prevalent neoantigen in colorectal,lung,and pancreatic cancers. TCR-T104 selectively recognizes and kills KRAS.G12V-expressing tumor cells. Combining T cell therapy with lymphodepleting chemotherapy significantly enhances tumor cell killing,particularly by TCR-T cells,tumor-infiltrating lymphocytes (TILs),and T cell engager antibodies across multiple cancer types and target antigens. Mechanistically,chemotherapy upregulates immunoproteasome activity and human leukocyte antigen (HLA)-I surface expression. HLA-immunopeptidome analyses reveal that chemotherapy remodels the antigenic landscape across tumor cell lines and in vivo models,increasing peptide abundance and hydrophobicity while altering proteasomal cleavage preferences. These findings establish a synergistic role for chemotherapy in enhancing neoantigen presentation and T cell-mediated tumor recognition and suggest that fine-tuning these regimens could improve ACT efficacy,particularly in tumors with low-abundance neoantigens. View Publication

Germinal center (GC) responses to T-dependent Ags require effective collaboration between Th cells,activated B cells,and follicular dendritic cells within a highly organized microenvironment. Studies using gene-targeted mice have highlighted nonredundant molecules that are key for initiating and maintaining the GC niche,including the molecules of the ICOS,CD40,and lymphotoxin (LT) pathways. Signaling through ICOS has multiple consequences,including cytokine production,expression of CD40L on Th cells,and differentiation into CXCR5(+) follicular Th cells,all of which are important in the GC reaction. We have therefore taken advantage of ICOS(-/-) mice to dissect which downstream elements are required to initiate the formation of GC. In the context of a T-dependent immune response,we found that GC B cells from ICOS(-/-) mice express lower levels of LTalphabeta compared with wild-type GC B cells in vivo,and stimulation of ICOS on T cells induces LTalphabeta on B cells in vitro. Administration of agonistic anti-LTbeta receptor Ab was unable to restore the GC response in ICOS(-/-) mice,suggesting that additional input from another pathway is required for optimal GC generation. In contrast,treatment with agonistic anti-CD40 Ab in vivo recovered GC networks and restored LTalphabeta expression on GC B cells in ICOS(-/-) mice,and this effect was dependent on LTbeta receptor signaling. Collectively,these data demonstrate that ICOS activation is a prerequisite for the up-regulation of LTalphabeta on GC B cells in vivo and provide a model for cooperation between ICOS,CD40,and LT pathways in the context of the GC response. View Publication

Understanding the cellular mechanisms that ensure an appropriate innate immune response against viral pathogens is an important challenge of biomedical research. In vitro studies have shown that natural killer (NK) cells purified from healthy donors can kill heterologous cell lines or autologous CD4+ T cell blasts exogenously infected with several strains of HIV-1. However,it is not known whether the deleterious effects of high HIV-1 viremia interferes with the NK cell-mediated cytolysis of autologous,endogenously HIV-1-infected CD4+ T cells. Here,we stimulate primary CD4+ T cells,purified ex vivo from HIV-1-infected viremic patients,with PHA and rIL2 (with or without rIL-7). This experimental procedure allows for the significant expansion and isolation of endogenously infected CD4+ T cell blasts detected by intracellular staining of p24 HIV-1 core antigen. We show that,subsequent to the selective down-modulation of MHC class-I (MHC-I) molecules,HIV-1-infected p24(pos) blasts become partially susceptible to lysis by rIL-2-activated NK cells,while uninfected p24(neg) blasts are spared from killing. This NK cell-mediated killing occurs mainly through the NKG2D activation pathway. However,the degree of NK cell cytolytic activity against autologous,endogenously HIV-1-infected CD4+ T cell blasts that down-modulate HLA-A and -B alleles and against heterologous MHC-I(neg) cell lines is particularly low. This phenomenon is associated with the defective surface expression and engagement of natural cytotoxicity receptors (NCRs) and with the high frequency of the anergic CD56(neg)/CD16(pos) subsets of highly dysfunctional NK cells from HIV-1-infected viremic patients. Collectively,our data demonstrate that the chronic viral replication of HIV-1 in infected individuals results in several phenotypic and functional aberrancies that interfere with the NK cell-mediated killing of autologous p24(pos) blasts derived from primary T cells. View Publication

OBJECTIVE: The T-cell receptor zeta (TCR zeta)-chain is a master sensor and regulator of lymphocyte responses. Loss of TCR zeta-chain expression has been documented during infectious and inflammatory diseases and defines a population of effector T cells (TCR zeta(dim) T cells) that migrate to inflamed tissues. We assessed the expression and functional correlates of circulating TCR zeta(dim) T cells in coronary artery disease. METHODS AND RESULTS: We examined the expression of TCR zeta-chain by flow cytometry in 140 subjects. Increased peripheral blood CD4(+) TCR zeta(dim) T cells were found in patients with acute coronary syndromes (ACS,n=66; median 5.3%,interquartile 2.6 to 9.1% of total CD4(+) T cells; Ptextless0.0001) compared to chronic stable angina (CSA,n=32; 1.6%; 1.0 to 4.1%) and controls (n=42; 1.5%; 0.5 to 2.9%). Such increase was significantly greater in ACS patients with elevated levels of C-reactive protein,and it persisted after the acute event. Moreover,TCR zeta(dim) cells were also more represented within CD8(+) T cell,NK,and CD4(+)CD28(null) T cell subsets in ACS compared to CSA and controls. Finally,CD4(+) and CD8(+) TCR zeta(dim) T cells isolated from ACS displayed an enhanced transendothelial migratory capacity. CONCLUSIONS: TCR zeta(dim) T cells,an effector T-cell subset with transendothelial migratory ability,are increased in ACS,and may be implicated in coronary instability. View Publication

The role of epitope-specific TCR repertoire diversity in the control of HIV-1 viremia is unknown. Further analysis at the clonotype level is important for understanding the structural aspects of the HIV-1 specific repertoire that directly relate to CTL function and ability to suppress viral replication. In this study,we performed in-depth analysis of T cell clonotypes directed against a dominantly recognized HLA B57-restricted epitope (KAFSPEVIPMF; KF11) and identified common usage of the TCR beta-chain TRBV7 in eight of nine HLA B57 subjects examined,regardless of HLA B57 subtype. Despite this convergent TCR gene usage,structural and functional assays demonstrated no substantial difference in functional or structural avidity between TRBV7 and non-TRBV7 clonotypes and this epitopic peptide. In a subject where TRBV7-usage did not confer cross-reactivity against the dominant autologous sequence variant,another circulating TCR clonotype was able to preferentially recognize the variant peptide. These data demonstrate that despite selective recruitment of TCR for a conserved epitope over the course of chronic HIV-1 infection,TCR repertoire diversity may benefit the host through the ability to recognize circulating epitope variants. View Publication