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The inability to scalably and precisely measure the activity of developmental cis-regulatory elements (CREs) in multicellular systems is a bottleneck in genomics. Here we develop a dual RNA cassette that decouples the detection and quantification tasks inherent to multiplex single-cell reporter assays. The resulting measurement of reporter expression is accurate over multiple orders of magnitude, with a precision approaching the limit set by Poisson counting noise. Together with RNA barcode stabilization via circularization, these scalable single-cell quantitative expression reporters provide high-contrast readouts, analogous to classic in situ assays but entirely from sequencing. Screening >200 regions of accessible chromatin in a multicellular in vitro model of early mammalian development, we identify 13 (8 previously uncharacterized) autonomous and cell-type-specific developmental CREs. We further demonstrate that chimeric CRE pairs generate cognate two-cell-type activity profiles and assess gain- and loss-of-function multicellular expression phenotypes from CRE variants with perturbed transcription factor binding sites. Single-cell quantitative expression reporters can be applied in developmental and multicellular systems to quantitatively characterize native, perturbed and synthetic CREs at scale, with high sensitivity and at single-cell resolution.

BACKGROUND: Combining cytotoxic chemotherapy or novel anticancer drugs with T-cell modulators holds great promise in treating advanced cancers. However, the response varies depending on the tumor immune microenvironment (TIME). Therefore, there is a clear need for pharmacologically tractable models of the TIME to dissect its influence on mono- and combination treatment response at the individual level. METHODS: Here we establish a patient-derived explant culture (PDEC) model of breast cancer, which retains the immune contexture of the primary tumor, recapitulating cytokine profiles and CD8+T cell cytotoxic activity. RESULTS: We explored the immunomodulatory action of a synthetic lethal BCL2 inhibitor venetoclax+metformin drug combination ex vivo, discovering metformin cannot overcome the lymphocyte-depleting action of venetoclax. Instead, metformin promotes dendritic cell maturation through inhibition of mitochondrial complex I, increasing their capacity to co-stimulate CD4+T cells and thus facilitating antitumor immunity. CONCLUSIONS: Our results establish PDECs as a feasible model to identify immunomodulatory functions of anticancer drugs in the context of patient-specific TIME.

Understanding the regulatory mechanisms facilitating hematopoietic stem cell (HSC) specification during embryogenesis is important for the generation of HSCs in vitro. Megakaryocyte emerged from the yolk sac and produce platelets, which are involved in multiple biological processes, such as preventing hemorrhage. However, whether megakaryocytes regulate HSC development in the embryonic aorta-gonad-mesonephros (AGM) region is unclear. Here, we use platelet factor 4 (PF4)-Cre;Rosa-tdTomato+ cells to report presence of megakaryocytes in the HSC developmental niche. Further, we use the PF4-Cre;Rosa-DTA (DTA) depletion model to reveal that megakaryocytes control HSC specification in the mouse embryos. Megakaryocyte deficiency blocks the generation and maturation of pre-HSCs and alters HSC activity at the AGM. Furthermore, megakaryocytes promote endothelial-to-hematopoietic transition in a OP9-DL1 coculture system. Single-cell RNA-sequencing identifies megakaryocytes positive for the cell surface marker CD226 as the subpopulation with highest potential in promoting the hemogenic fate of endothelial cells by secreting TNFSF14. In line, TNFSF14 treatment rescues hematopoietic cell function in megakaryocyte-depleted cocultures. Taken together, megakaryocytes promote production and maturation of pre-HSCs, acting as a critical microenvironmental control factor during embryonic hematopoiesis.

INTRODUCTION: SARS-CoV-2 infection increases the risk of worse outcomes in cancer patients, including those with breast cancer. Our previous study reported that the SARS-CoV-2 membrane protein (M-protein) promotes the malignant transformation of triple-negative breast cancer cells (triple-negative BCC). METHODS: In the present study, the effects of M-protein on the ability of extracellular vesicles (EV) derived from triple-negative BCC to regulate the functions of tissue stem cells facilitating the tumor microenvironment were examined. RESULTS: Our results showed that EV derived from M-protein-induced triple-negative BCC (MpEV) significantly induced the paracrine effects of adipose tissue-derived mesenchymal stem cells (ATMSC) on non-aggressive BCC, promoting the migration, stemness phenotypes, and in vivo metastasis of BCC, which is related to PGE2/IL1 signaling pathways, in comparison to EV derived from normal triple-negative BCC (nEV). In addition to ATMSC, the effects of MpEV on endothelial progenitor cells (EPC), another type of tissue stem cells, were examined. Our data suggested that EPC uptaking MpEV acquired a tumor endothelial cell-like phenotype, with increasing angiogenesis and the ability to support the aggressiveness and metastasis of non-aggressive BCC. DISCUSSION: Taken together, our findings suggest the role of SARS-CoV-2 M-protein in altering the cellular communication between cancer cells and other non-cancer cells inside the tumor microenvironment via EV. Specifically, M-proteins induced the ability of EV derived from triple-negative BCC to promote the functions of non-cancer cells, such as tissue stem cells, in tumorigenesis.

Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disease with a clear genetic component. While most SLE patients carry rare gene variants in lupus risk genes, little is known about their contribution to disease pathogenesis. Amongst them, SH2B3-a negative regulator of cytokine and growth factor receptor signaling-harbors rare coding variants in over 5% of SLE patients. Here, we show that unlike the variant found exclusively in healthy controls, SH2B3 rare variants found in lupus patients are predominantly hypomorphic alleles, failing to suppress IFNGR signaling via JAK2-STAT1. The generation of two mouse lines carrying patients' variants revealed that SH2B3 is important in limiting the number of immature and transitional B cells. Furthermore, hypomorphic SH2B3 was shown to impair the negative selection of immature/transitional self-reactive B cells and accelerate autoimmunity in sensitized mice, at least in part due to increased IL-4R signaling and BAFF-R expression. This work identifies a previously unappreciated role for SH2B3 in human B cell tolerance and lupus risk.

BACKGROUND: There is little information on the trajectory and developmental fate of Lin-CD34+DNAM-1bright CXCR4+ progenitors exiting bone marrow during systemic inflammation. OBJECTIVE: To study Lin-CD34+DNAM-1bright CXCR4+ cell circulation in cancer patients, to characterize their entry into involved lung tissue and to characterize their progenies. METHODS: Flow cytometric analysis of PBMC from 18 patients with lung cancer on samples collected immediately before the first and the second treatment was performed to study Lin-CD34+DNAM-1bright CXCR4+ precursors. Precursors were purified (>99%) and cultured in vitro from all patients. Paired PBMC and tissue samples from patients undergoing tumor resection were analyzed by flow cytometry to assess tissue entry and compare phenotype and developmental potential of Lin-CD34+DNAM-1bright CXCR4+ cells in both compartments. RESULTS: Significant circulation of Lin-CD34+DNAM-1bright CXCR4+ precursors was observed 20d after the first treatment. Precursors express CXC3CR1, CXCR3, CXCR1 consistent with travel towards inflamed tissues. Flowcytometric analysis of lung tissue samples showed precursor presence in all patients in tumor and neighboring uninvolved areas. Successful purification and in vitro culture from both blood and lung tissue generates a minor proportion of maturing NK cells (<10%) and a predominant proportion (>85%) of α/β T-progenies with innate-like phenotype expressing NKG2D,NKp30,DNAM-1. Innate-like maturing T-cells in vitro are cytotoxic, can be triggered via NKR/TCR co-stimulation and display broad spectrum Th1,Th2 and Th1/Th17 cytokine production. CONCLUSION: In advanced stage lung cancer CD34+DNAM-1brightCXCR4+ inflammatory precursors increase upon treatment, enter involved tissues, generate functional progenies and may thus represent an additional player contributing to immune balance in the highly SDF-1/CXCR4-biased pro-metastatic tumor microenvironment.

Vascular disruption has been implicated in coronavirus disease 2019 (COVID-19) pathogenesis and may predispose to the neurological sequelae associated with long COVID, yet it is unclear how blood-brain barrier (BBB) function is affected in these conditions. Here we show that BBB disruption is evident during acute infection and in patients with long COVID with cognitive impairment, commonly referred to as brain fog. Using dynamic contrast-enhanced magnetic resonance imaging, we show BBB disruption in patients with long COVID-associated brain fog. Transcriptomic analysis of peripheral blood mononuclear cells revealed dysregulation of the coagulation system and a dampened adaptive immune response in individuals with brain fog. Accordingly, peripheral blood mononuclear cells showed increased adhesion to human brain endothelial cells in vitro, while exposure of brain endothelial cells to serum from patients with long COVID induced expression of inflammatory markers. Together, our data suggest that sustained systemic inflammation and persistent localized BBB dysfunction is a key feature of long COVID-associated brain fog.

The respiratory mucosal system plays a critical role in the pathogenesis of allergic asthma (AA). Currently, therapeutic Fc fusion proteins are as a promising strategy for mucosal vaccine delivery systems. In this work, a plasmid encoding the Mycobacterium tuberculosis ESAT6-Fc fusion protein was successfully constructed, and high-purity ESAT6-Fc fusion protein was subsequently obtained. Administered via intranasal immunization in OVA-induced allergic asthma model mice, ESAT6-Fc fusion protein significantly alleviated airway inflammation and mucus production, and reduced the proportions of Th2 cells, Th17 cells, and eosinophils, while increasing the proportions of Th1 cells with no histopathological changes to major organs. To elucidate the underlying immune regulatory mechanisms of ESAT6, integrated transcriptomic and proteomic analyses were performed, revealing Th1/Th2 cell differentiation and Th17 cell differentiation as the two most significantly enriched pathways at both the gene and protein levels. CD3e (CD3E) and CD3g (CD3G), two essential subunits of the TCR–CD3 complex, were identified as core target factors. The validations from the ESAT6-Fc-treated AA lung tissues, as well as co-cultured TH0 cells from C57BL/6J mice and CD2.4 dendritic cells exposed to the ESAT6-Fc protein, were consistent with the aforementioned findings. ESAT6-Fc exhibits a safe profile with favorable efficacy against OVA-induced AA via intranasal immunization, and ESAT6 ameliorates AA by regulating the differentiation of Th0 cells into Th1 cells, which were closely associated with the down-regulation of CD3e and CD3g expression, presumably leading to the impairment of TCR–CD3 complex assembly. ESAT6-Fc fusion protein demonstrates promise as a potential safe intranasal immunotherapy agent for the treatment of AA.

AbstractBackground Chimeric antigen receptor (CAR) T-cell therapies are highly efficacious for several different hematologic cancers. However, for most CAR T targets it is observed that low surface antigen density on tumors can significantly reduce therapeutic efficacy. In this study, we explore this dynamic in the context of CD72, a surface antigen we recently found as a promising target for refractory B-cell cancers, but for which CD72 low antigen density can lead to therapeutic resistance in preclinical models.MethodsPrimary samples were accessed via institutional review board-approved protocols. Affinity-matured and humanized nanobody clones were previously described in Temple et al. (2023). CAR T cells were generated via lentiviral transduction. In vitro cytotoxicity assays were performed using luciferase-labeled cell lines. In vivo studies were performed using cell line-derived or patient-derived xenografts implanted in NOD scid gamma mice.ResultsWe first confirmed ubiquitous CD72 expression across a range of primary B-cell non-Hodgkin lymphomas. We further found that after resistance to CD19-directed therapies, across both B-cell acute lymphoblastic leukemia (B-ALL) models and primary tumor samples, surface CD72 expression was largely preserved while CD22 expression was significantly diminished. Affinity maturation of a nanobody targeting CD72, when incorporated into CAR T cells, led to more effective elimination in vitro of isogenic models of CD72 low-expressing tumors. These results suggested that nanobody-based CAR T cells (nanoCARs) may exhibit a similar relationship between binder affinity, antigen expression, and efficacy as previously demonstrated only for single chain variable fragment-based CAR T cells. Surprisingly, however, this significantly improved in vitro efficacy only translated to modest in vivo survival benefit. As a parallel strategy to enhance CAR T function, we found that the small molecule bryostatin could also significantly increase CD72 surface antigen density on B-cell malignancy models. Structural modeling and biochemical analysis identified critical residues improving CD72 antigen recognition of our lead affinity-matured nanobody.ConclusionsTogether, these findings support affinity-matured CD72 nanoCARs as a potential immunotherapy product for CD19-refractory B-cell cancers. Our results also suggest that for B-ALL in particular, CD72 may be a preferable second-line immunotherapy target over CD22.

Purposers12979860 polymorphism of the lambda 4 interferon (IFNL4) gene has been related with Hepatitis C Virus (HCV) spontaneous clearance (CC genotype favourable versus CT/TT). The implications of this polymorphism over chronic-HCV disease are unknown, particularly during Human Immunodeficiency virus (HIV) coinfection.MethodsObservational study in 118 people with HIV (PWH): 45 with HCV-chronic infection (CHC); 38 who spontaneously clear HCV (SC) and 35 never infected by HCV (HIV). Expression of surface markers was evaluated in CD4+ and CD8+ memory T cells by flow cytometry, and plasma markers were quantified by Luminex or ELISA.ResultsCHC individuals with CC genotype (CHC-CC) showed higher expression of markers suggestive of senescence (PD1/CD57) and activation markers (CD38/CD25) in CD8 + T-cell subpopulations, compared to CT/TT carriers. Similarly, significantly higher expression was observed in CD4+ and CD8+ T cells in CHC-CC than SC-CC and HIV-CC controls. Regarding plasma markers, CHC-CC group had higher levels of 19 plasma immune biomarkers compared to CT/TT carriers, and 16 relative to SC-CC, including pro- and anti-inflammatory cytokines, while oxidative stress slightly diminished in CHC-CC individuals.ConclusionsCC-carriage may result in premature cellular activation and senescence or impaired functionality during HCV chronic infection in PWH, which may be masked by their maintained plasma immune homeostasis during clinical monitoring.Graphical Abstract Supplementary InformationThe online version contains supplementary material available at 10.1186/s12967-025-07070-5.

Multiple sclerosis (MS), the most prevalent chronic inflammatory, demyelinating and neurodegenerative disease of the central nervous system in young adults, exhibits marked sexual dimorphism, with a 3:1 female-to-male ratio, but more severe symptoms and greater neurological damage in males. Increasing attention has focused on identifying circulating molecules that reflect inflammatory activity within the central nervous system and could clarify the mechanisms underlying MS. Pleiotrophin (PTN), a cytokine implicated in autoimmune and neurological diseases, is significantly elevated in patients with relapsing-remitting MS (RRMS). To explore the potential contribution of PTN and its receptors to neuroinflammatory signaling, we quantified the mRNA expression of PTN receptors in peripheral blood mononuclear cells from RRMS patients compared to untreated RRMS patients and healthy control subjects. We further performed an in silico molecular docking and molecular dynamics analysis to assess the possible functional significance of PTN-receptor interactions. Our results show a significant overexpression of integrin subunit beta-3 (ITGB3) mRNA in peripheral blood mononuclear cells from RRMS patients compared to healthy control subjects. Molecular docking shows that PTN could binds to the metal ion-dependent adhesion site domain of ITGB3 via Mg2+/Ca2+-mediated stabilization and has a higher binding affinity than fibrinogen, the canonical endogenous ligand. These findings suggest that ITGB3 could be a dynamically regulated integrin receptor in RRMS that may participate in PTN-driven neuroinflammatory pathways in peripheral blood immune cells, influenced by disease stage, sex, and immunotherapy. While our results support the biological plausibility of PTN–ITGB3 engagement, they remain hypothesis-generating and require functional validation. The integration of molecular expression data and computational modeling underscores the potential involvement of ITGB3 as a possible participant in MS and warrants further investigation of its clinical and mechanistic role.

Simple SummaryDespite effective therapies, multiple myeloma remains an incurable disease. Even when achieving deep remissions, almost all patients eventually relapse. Current evaluation of this persistent disease is based on bone marrow evaluation, but this approach has significant drawbacks. Blood-based disease evaluation is less invasive and potentially more comprehensive in evaluation of total tumor mass but lacks sensitivity. It is currently unknown which biomarker in blood is superior. Therefore, we tested three different blood-derived DNA sources: peripheral blood mononuclear cells, enriched circulating tumor cells (CTCs) and cell-free DNA. Enrichment of CTCs, followed by next-generation sequencing, resulted in the highest sensitivity. In patients with detectable disease in their bone marrow, but no detectable CTCs, the first relapse occurred after almost 4 years, while early relapse (<18 months) occurred in 5/12 patients with detectable CTCs. AbstractBackground/Objectives: Minimal residual disease (MRD) persists in most multiple myeloma (MM) patients, causing relapse despite deep remissions. Repeatability of MRD detection in MM bone marrow (BM) samples is limited, underscoring the need for blood-based monitoring approaches that can allow more thorough disease surveillance. Methods: This study compares tumor detection rates in BM-derived DNA with different blood-derived DNA sources, using next-generation sequencing of the immunoglobulin locus (NGS-IG). CD138-targeted immunomagnetic enrichment of circulating tumor cells (CTCs) followed by vacuum evaporation to concentrate DNA was used to optimize the tumor detection rate. Results: Tumor DNA was detected in 76%, 88% and 100% of cell-free DNA, peripheral blood-derived mononuclear cells, DNA and enriched CTC-DNA samples of patients with active myeloma, respectively. These data indicate that enriched CTC samples were the most informative for evaluation of disease detection with NGS-IG in patients with active myeloma. MRD detection was performed in paired BM and enriched CTC-DNA samples from 37 patients in remission. MRD positivity was found in the BM of 24 patients, with half of them (12/24) also showing the presence of MRD when enriched CTC-DNA was used. Interestingly, time to progression (TTP) of enriched CTC MRD-negative/BM MRD-positive patients was comparable to that of double MRD-negative (BM and CTC) patients. Moreover, double positive patients showed a trend to earlier relapses. Conclusions: Our data suggest that NGS-IG analysis with enriched CTC-DNA may offer improved predictive abilities for relapse in multiple myeloma compared to the currently used BM-DNA-based tumor detection method.