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Objective: To develop a rapid functional assay to validate variants of uncertain significance (VUS) in the MEFV gene. Methods: Overactivity of the pyrin inflammasome pathway and ASC speck oligomerisation in response to stimulation with low concentrations of Clostridium difficile toxin A was directly visualised by immunofluorescence microscopy. A semi‐automated algorithm was developed to count cells and ASC specks. Results: The semi‐automated ASC speck assay is able to discriminate between healthy controls and patients with familial Mediterranean fever (FMF) and pyrin inflammasome overactivity with high sensitivity. It is also able to discriminate pyrin inflammasome overactivity from other autoinflammatory disease controls with high specificity. Conclusion: The semi‐automated ASC speck assay may be a useful test to functionally validate VUS in the MEFV gene and screen for pyrin inflammasome overactivity. A semi‐automated ASC speck assay using machine learning is able to discriminate between healthy controls and patients with familial Mediterranean fever (FMF) with high sensitivity. It is also able to discriminate FMF from other autoinflammatory diseases with high specificity.

Aggressive non‐Hodgkin lymphoma (aNHL) often relapses after first‐line treatment. Clinical data supports the safety and efficacy of the combination of mosunetuzumab, a CD20×CD3 bispecific antibody, and polatuzumab vedotin, an anti‐CD79b antibody drug conjugate (Mosun‐Pola) in relapsed/refractory aNHL. This study investigated the molecular mechanism behind the combination effect of Mosun‐Pola in human diffuse large B‐cell lymphoma (DLBCL) cell lines. Methods: The in vitro Mosun‐Pola efficacy in DLBCL cells (SU‐DHL‐8 and HT) was evaluated by T cell‐dependent cellular cytotoxicity (TDCC) assay. CD20‐stable‐knockdown SU‐DHL‐8 cells were established using lentiviral short hairpin RNA. Surface and T‐cell activation marker proteins expression were determined by flow cytometry. Human T‐cell‐injected mice or humanized NOD/Shi‐scid, IL‐2Rγnull (huNOG) mice were used for an in vivo study. Results: An in vitro TDCC assay showed a synergistic effect in SU‐DHL‐8 and HT cells. Based on our experimental results of suppressing CD20 expression, it was suggested that this combination effect could be caused by an increase in CD20 expression by polatuzumab vedotin. In addition, examining the effects of CD20 upregulation in tumor cells on T‐cell activation demonstrated that the combination of Mosun‐Pola enhanced T‐cell activation markers in both CD4+ and CD8+ T cells during the TDCC reaction. In vivo studies, using human immune system‐reconstituted mouse models confirmed that polatuzumab vedotin enhanced CD20 expression in tumors, and the combination of Mosun‐Pola showed significantly improved anti‐tumor effects compared with single‐drug treatments. Conclusions: These findings suggest that polatuzumab vedotin‐induced CD20 upregulation provides a molecular rationale to explain the synergistic effect of this combination therapy.Trial RegistrationThe authors have confirmed clinical trial registration is not needed for this submission.

Tolerogenic dendritic cells (tolDCs) that dampen T cell responses can be induced from blood monocytes in vitro using factors such as Vitamin D3 (VitD), dexamethasone, IL‐10, or rapamycin. However, challenges remain in obtaining robust and efficient generation of cell therapy‐based tolDCs without compromising their viability. We recently reported that CCR2‐dependent recruitment of monocytic cells, with the capacity to dampen T‐helper responses, occurs in mice treated with a single‐stranded oligonucleotide (ssON). Here, we investigated the effects of this immunomodulatory noncoding ssON on differentiating human monocytes towards DC in the presence of IL‐4 and GM‐CSF (moDC). The moDC differentiated in the presence of ssON upregulated CD1a but also increased their expression of PD‐L1. The differentiation of monocytes to moDC in the presence of ssON introduced transcriptomic changes, many of which overlapped with VitD‐moDC and resulted in moDCs with altered lipopolysaccharide (LPS)‐responsiveness. Moreover, ssON‐moDC exhibited a low capacity to stimulate alloreactive T cells in vitro and instead promoted the induction of CD4+FoxP3+CD25+ T cells. Experiments using chemical reagents support a role for PPAR‐γ in the generation of ssON‐moDC. Collectively, our data show that monocytes differentiated with IL‐4, GM‐CSF, and ssON generate cells with phenotypic and functional characteristics of tolDCs. In this article, the authors elucidated the immunoregulatory role of an oligonucleotide (ssON) that favors the induction of human tolerogenic dendritic cells (DC). The tolerogenic profile was evidenced by reduced responsiveness to lipopolysaccharides (LPS) (A). Importantly, the tolerogenic DCs had upregulated PD‐L1 molecules and functionally inhibited the proliferation of alloreactive T cells and induced FoxP3+ Tregs (B). This study envisions the development of ssON as therapeutic for rebalancing overactive T‐helper cell responses.

Ataxia with oculomotor apraxia type 1 (AOA1) is a rare, autosomal recessive, early-onset, progressive cerebellar ataxia caused by mutations in the APTX gene, which encodes aprataxin, a DNA-adenylate hydrolase involved in DNA damage repair. The pathogenesis of AOA1 remains unclear. The purpose of this study was to investigate the pathogenesis of a novel mutation, p.H201P/H201R, carried by our AOA1 patient and the mechanism of AOA1 in an induced pluripotent stem cells (iPSCs) model. We edited iPSCs derived from a healthy individual to carry the APTX homozygous mutation p.H201P (H201P-iPSCs) or p.H201R (H201R-iPSCs) via CRISPR/Cas9. We found that aprataxin expression was absent in both H201P- and H201R-iPSCs. The capacity of these APTX-mutant iPSCs to differentiate into neural progenitor cells (NPCs) and mature neurons was diminished. We observed an increase in DNA single-strand breaks (SSB) via a comet assay and poly(ADP-ribose) staining, and an increase in the ratio of cleaved PARP-1/total PARP-1 in APTX-mutant NPCs and early immature neurons (EiNs), in addition of a heightened sensitivity to tert-butyl hydroperoxide in APTX-mutant EiNs. Moreover, a decrease of APE1 expression was observed in APTX-mutant NPCs and H201R-EiNs during neural differentiation. Our study established a practical iPSCs model to investigate AOA1 disease. We found that mutant aprataxin leads to defective neural differentiation, accompanied by the accumulation of DNA SSBs with increased cleaved PARP-1 and reduced APE1 expression of the base excision repair pathway.

Chagas disease, caused by the parasite Trypanosoma cruzi, affects millions of people globally. Unfortunately, the available treatment options, especially for the chronic stage of the disease, are suboptimal. Given the chronic nature of the disease and the elusive nature of the parasite, there is a high need for new and safer drugs that deliver sterile cure. Posaconazole was a promising lead in the drug discovery pipeline but ultimately failed in clinical trials due to patient relapses. This failure illustrates the need for a drug screening assay that can predict sterile cure by assessing recrudescence after treatment. Here, we used human induced pluripotent stem cell (iPSC)-derived macrophages (iMACs) as host cells for T. cruzi. The iMACs were highly susceptible to infection by the parasites. By combining red fluorescent protein (RFP)-expressing iMACs with mNeonGreen-expressing T. cruzi, we were able to monitor the dynamics of the infection through live cell imaging. The activity of the compounds benznidazole and posaconazole was consistent with the results of an established infection system using mouse primary macrophages. The post-mitotic nature of iMACs makes them suitable host cells for long-term assays needed to assess recrudescence of parasites. Moreover, their human origin, stable genetic background, and capacity for genetic modification make the iMACs excellent host cells for studying host-pathogen interaction. Author summaryThe parasite Trypanosoma cruzi, the causative agent of Chagas disease, is a global health concern affecting millions each year. Infection with T. cruzi can cause chronic disease, often remaining asymptomatic for decades before resulting in severe cardiac or gastro-intestinal pathologies. To date, only benznidazole and nifurtimox are used for treatment of the infection, but both drugs are suboptimal for curing the chronic stage. Posaconazole showed great promise in preclinical studies but failed to achieve sterile cure in clinical trials, causing patient relapses. These disappointing results underline the need for drug screening assays able to predict sterile cure by evaluating recrudescence post-treatment. We used human induced pluripotent stem cell derived macrophages as host cells for T. cruzi and testing of trypanocidal compounds. This model can be used for long-term in vitro screening assays to find new drug candidates against Chagas disease. The human origin of these cells combined with the possibility of upscaling their production make them great host cells for drug screening campaigns.

In stem cell biology, a long-held structure–function relationship is the domed colony morphology and naïve pluripotency for mouse or human pluripotent stem cells. This link has provided a convenient way to recognize bona fide naïve pluripotent cells during derivation, passaging and characterization. However, the molecular basis of this link remains poorly understood. Results: We show that a loss of domed morphology may not impact the overall genetic architecture of naïve pluripotency in mouse embryonic stem cells (mESCs). We first generated stable mESC lines by knocking out Myh9 that encodes non-muscle myosin heavy chain IIA, resulting in colonies deprived of the typical domed morphology, but competent to differentiate into the three germ layers and chimeric mice. Modulating cell morphologies with inhibitors against kinases known to regulate myosin pathway also phenocopy the knockout in wild type mESCs. Conclusions: These results provide evidence that the domed morphology and potency can be uncoupled and suggest that domed structure is not a pre-requisite for acquiring and maintaining naïve pluripotency.

EVI1-rearranged acute myeloid leukemia (AML) with inv(3)(q21q26) or t(3;3)(q21q26) represents a distinct and aggressive subtype characterized by poor prognosis and limited treatment options. However, the optimal strategy to overcome resistance to conventional therapy remains elusive. Building upon observations correlating EVI1 overexpression with reduced sensitivity to venetoclax, a BH3-mimetic BCL-2 inhibitor, we investigated the mechanisms of resistance to venetoclax in combination with hypomethylating agents in inv(3)/t(3;3) AML cells. Utilizing novel murine models recapitulating inv(3) AML with concomitant SF3B1 mutations, we conducted comprehensive phenotypic and transcriptomic analyses in the presence or absence of venetoclax-containing therapy. Despite initial therapeutic responses, manifested as partially prolonged survival and myeloid differentiation, resistant leukemic cells demonstrated enhanced dependency on BRD4 and MYB pathways with a dormant phenotype. Notably, inhibition of either BRD4 or MYB significantly augmented the efficacy of venetoclax and hypomethylating agents in both murine and patient-derived AML models harboring inv(3) and SF3B1 mutations. These findings elucidate the transcriptional dynamics underlying venetoclax resistance and propose alternative therapeutic strategies targeting BRD4 and MYB as promising avenues for improving outcomes in patients with EVI1-rearranged AML. Our work highlights the necessity for innovative combination therapies to address the multifaceted mechanisms of resistance in this high-risk leukemia subtype.

Chimeric antigen receptor (CAR) T cell therapy has transformed the treatment landscape of hematologic cancers by engineering T cells to specifically target and destroy cancer cells. Monitoring CAR T cell activity and function is essential for optimizing therapeutic outcomes, but existing tools for CAR detection are often limited in specificity and functional assessment capability. Methods: We developed dextran multimers by conjugating multiple CAR-specific antigens to a dextran backbone. The multimers were compared to previously reported antigen tetramers for their ability to stain and detect CAR T cells. Because these multimers incorporate the CAR target antigen, they uniquely enable assessment of CAR T cell functionality. We tested the staining and functional properties of the multimers across a range of CAR constructs with different affinities, using flow cytometry and microscopy. Results: The dextran multimers demonstrated high specificity and sensitivity in staining CAR T cells, with adjustable antigen density to optimize binding. Dextran multimers also enabled effective clustering and subsequent activation of CARs, showing their utility as both a staining and functional assessment tool. The multimers revealed that CARs with different affinities and clustering tendencies displayed varied binding and activation in response to different antigen densities. Conclusions: Dextran multimers offer a dual advantage as versatile reagents for both staining and functional analysis of CAR T cells. Their capacity to engage CARs with the specific antigen provides a valuable platform for evaluating CAR functionality, informing CAR design improvements, and enhancing therapeutic precision.