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This work demonstrates that group 2 KLF family transcription factors are critical for specifying the identity of distinct tissue-resident macrophages. KLF2 directly controls expression of genes previously shown to be necessary in cavity macrophages, while KLF4 may play a similar role in alveolar macrophages. Tissue-resident macrophages adopt distinct gene expression profiles and exhibit functional specialization based on their tissue of residence. Recent studies have begun to define the signals and transcription factors that induce these identities. Here we describe an unexpected and specific role for the broadly expressed transcription factor Krüppel-like factor 2 (KLF2) in the development of embryonically derived large cavity macrophages (LCMs) in the serous cavities. KLF2 not only directly regulates the transcription of genes previously shown to specify LCM identity, such as retinoic acid receptors and GATA6, but also is required for induction of many other transcripts that define the identity of these cells. Our results suggest that KLF4 may similarly regulate the identity of alveolar macrophages in the lung. These data demonstrate that broadly expressed transcription factors, such as group 2 KLFs, can play important roles in the specification of distinct identities of tissue-resident macrophages.

ABSTRACTBacteriophages (phages) are emerging as a viable adjunct to antibiotics for the treatment of multidrug‐resistant (MDR) bacterial infections. While intravenous phage therapy has proven successful in many cases, clinical outcomes remain uncertain due to a limited understanding of host response to phages. In this study, we conducted a comprehensive examination of the interaction between clinical‐grade phages used to treat MDR Escherichia coli and Klebsiella pneumoniae infections, and human peripheral blood immune cells. Using whole transcriptome as well as proteomic approaches, we identified a strong inflammatory response to E. coli phage vB_EcoM‐JIPh_Ec70 (herein, JIPh_Ec70) that was absent upon exposure to K. pneumoniae phage JIPh_Kp127. We confirmed that JIPh_Ec70's DNA recognition by the STING pathway was principally responsible for the activation of NF‐kB and the subsequent inflammatory response. We further show that monocytes and neutrophils play a dominant role in phage uptake, primarily through complement‐mediated phagocytosis. Significant differences in complement‐mediated phagocytosis of JIPh_Kp127 and JIPh_Ec70 were observed, suggesting that reduced recognition, phagocytosis, and immunogenicity all contribute to the significantly decreased response to JIPh_Kp127. Our findings contribute to the progress of our understanding of the innate immune response to therapeutic phages and offer potential insights into how to improve the safety and effectiveness of phage therapy. Clinical grade JIPh_Ec70 phages but not JIPh_Kp127 phages elicit a potent inflammatory response in peripheral immune cells. JIPh_Ec70 phagocytic engulfment is facilitated by complement opsonization, resulting in STING activation by phage DNA, driving an inflammatory signaling cascade. Understanding phage immunogenicity will be a key factor in developing effective phage therapies in the coming years.

Uveal melanoma (UM) is the most common intraocular cancer in adults, with metastatic disease (mUM) occurring in approximately half of the patients. Tebentafusp, an immune-mobilizing monoclonal T cell receptor against cancer (ImmTAC), is a therapeutic shown to improve overall survival (OS) in HLA-A*02:01+ adult patients with mUM. Here we investigate the impact of tumor-associated macrophages (TAM) on ImmTAC activity. In vitro, M2 macrophages inhibit ImmTAC-mediated tumor-killing in a dose-dependent and contact-dependent manner. Accordingly, high baseline intratumoral TAM-to-T cell ratios correlate with shorter OS (HR = 2.09, 95% CI, 1.31–3.33, p = 0.002) in tebentafusp-treated mUM patients from a phase 2 trial. By contrast, IL-2 conditioning of T cells overcomes M2 macrophage-mediated suppression in vitro, while ImmTAC treatment leads to M2-to-M1 macrophage reprogramming both in vitro and in tebentafusp-treated mUM patients. Overall, we show that tebentafusp reshapes the tumor microenvironment to enhance anti-tumor T cell activity, whilst combining tebentafusp with IL-2 may enhance benefit in patients with high levels of TAM. ‘T cell engagers promote antitumor immunity, but how macrophage modulates this activity in tumor is still unclear. Here the authors show, using biopsies from patients with uveal melanoma and single cell analyses, that a T cell engager, tebentafusp, reprograms tumor-associated macrophages and ameliorates, in synergy with IL-2, immunosuppression to cancer.

The levels of transcription factor Ets1 are high in resting B and T cells, but are downregulated by signaling through antigen receptors and Toll-like receptors (TLRs). Loss of Ets1 in mice leads to excessive immune cell activation and development of an autoimmune syndrome and reduced Ets1 expression has been observed in human PBMCs in the context of autoimmune diseases. In B cells, Ets1 serves to prevent premature activation and differentiation to antibody-secreting cells. Given these important roles for Ets1 in the immune response, stringent control of Ets1 gene expression levels is required for homeostasis. However, the genetic regulatory elements that control expression of the Ets1 gene remain relatively unknown. Here we identify a topologically-associating domain (TAD) in the chromatin of B cells that includes the mouse Ets1 gene locus and describe an interaction hub that extends over 100 kb upstream and into the gene body. Additionally, we compile epigenetic datasets to find several putative regulatory elements within the interaction hub by identifying regions of high DNA accessibility and enrichment of active enhancer histone marks. Using reporter constructs, we determine that DNA sequences within this interaction hub are sufficient to direct reporter gene expression in lymphoid tissues of transgenic mice. Further analysis indicates that the reporter construct drives faithful expression of the reporter gene in mouse B cells, but variegated expression in T cells, suggesting the existence of T cell regulatory elements outside this region. To investigate how the downregulation of Ets1 transcription is associated with alterations in the epigenetic landscape of stimulated B cells, we performed ATAC-seq in resting and BCR-stimulated primary B cells and identified four regions within and upstream of the Ets1 locus that undergo changes in chromatin accessibility that correlate to Ets1 gene expression. Interestingly, functional analysis of several putative Ets1 regulatory elements using luciferase constructs suggested a high level of functional redundancy. Taken together our studies reveal a complex network of regulatory elements and transcription factors that coordinate the B cell-specific expression of Ets1.

Immune evasion by cancer cells involves reshaping the tumor microenvironment (TME) via communication with non-malignant cells. However, resistance-promoting interactions during treatment remain lesser known. Here we examine the composition, communication, and phenotypes of tumor-associated cells in serial biopsies from stage II and III high-risk estrogen receptor positive (ER+ ) breast cancers of patients receiving endocrine therapy (letrozole) as single agent or in combination with ribociclib, a CDK4/6-targeting cell cycle inhibitor. Single-cell RNA sequencing analyses on longitudinally collected samples show that in tumors overcoming the growth suppressive effects of ribociclib, first cancer cells upregulate cytokines and growth factors that stimulate immune-suppressive myeloid differentiation, resulting in reduced myeloid cell- CD8 + T-cell crosstalk via IL-15/18 signaling. Subsequently, tumors growing during treatment show diminished T-cell activation and recruitment. In vitro, ribociclib does not only inhibit cancer cell growth but also T cell proliferation and activation upon co-culturing. Exogenous IL-15 improves CDK4/6 inhibitor efficacy by augmenting T-cell proliferation and cancer cell killing by T cells. In summary, response to ribociclib in stage II and III high-risk ER +  breast cancer depends on the composition, activation phenotypes and communication network of immune cells. The CDK4/6 inhibitor ribociclib holds promise in cancer therapy but how cell cycle inhibitory drugs affect the anti-tumor immune response remains a question. Here authors show that poor response of early-stage estrogen receptor positive breast cancers to ribociclib is caused by changes in the immune cell composition and cancer-cell-immune-cell communication in the tumors rather than intrinsic cancer cell resistance to cell cycle inhibition.

AbstractBackgroundHypoxia is associated with the evasion of triple-negative breast cancer (TNBC) from immune surveillance. Hypoxia increases the subpopulation of putative TNBC stem-like cells (TNBCSCs) through activating Wnt/β-Catenin signaling. The shedding of MHC class I-related chain A (MICA) is particularly noteworthy in cancer stem cells (CSCs), promoting the resistance of CSCs to natural killer (NK) cell cytotoxicity. To reestablish MICA/NKG2D-mediated immunosurveillance, we proposed the design of a fusion protein (SHH002-hu1-MICA) which consists of Frizzled-7 (Fzd7)-targeting antibody and MICA, serving as an engager retargeting NK cells against TNBCs, especially TNBCSCs.MethodsOpal multicolor immunohistochemistry staining was used to validate the expression of membrane MICA (mMICA) and existence of NK cells in TNBC tumors; flow cytometry (FCM) assay was used to detect the expression of Fzd7/mMICA on TNBCs. Biolayer interferometry (BLI) and surface plasmon resonance (SPR) assays were executed to assess the affinity of SHH002-hu1-MICA towards rhFzd7/rhNKG2D; near-infrared imaging assay was used to evaluate the targeting capability. A cytotoxicity assay was conducted to assess the effects of SHH002-hu1-MICA on NK cell-mediated killing of TNBCs, and FCM assay to analyze the effects of SHH002-hu1-MICA on the degranulation of NK cells. Finally, TNBC cell-line-derived xenografts were established to evaluate the anti-tumor activities of SHH002-hu1-MICA in vivo.ResultsThe expression of mMICA is significantly downregulated in hypoxic TNBCs and TNBCSCs, leading to the evasion of immune surveillance exerted by NK cells. The expression of Fzd7 is significantly upregulated in TNBCSCs and exhibits a negative correlation with the expression of mMICA and infiltration level of NK cells. On accurate assembly, SHH002-hu1-MICA shows a strong affinity for rhFzd7/rhNKG2D, specifically targets TNBC tumor tissues, and disrupts Wnt/β-Catenin signaling. SHH002-hu1-MICA significantly enhances the cytotoxicity of NK cells against hypoxic TNBCs and TNBCSCs by inducing the degranulation of NK cells and promotes the infiltration of NK cells in CD44high regions within TNBC xenograft tumors, exhibiting superior anti-tumor activities than SHH002-hu1.ConclusionsSHH002-hu1-MICA maintains the targeting property of SHH002-hu1, successfully activates and retargets NK cells against TNBCs, especially TNBCSCs, exhibiting superior antitumor activities than SHH002-hu1. SHH002-hu1-MICA represents a promising new engager for NK cell-based immunotherapy for TNBC.

BackgroundMacrophages are required for development and tissue repair and protect against microbial attacks. In response to external signals, monocytes differentiate into macrophages, but our knowledge of changes that promote this transition at the level of mRNA processing, in particular mRNA polyadenylation, needs advancement if it is to inform new disease treatments. Here, we identify CFIm25, a well-documented regulator of poly(A) site choice, as a novel mediator of macrophage differentiation.MethodsCFIm25 expression was analyzed in differentiating primary human monocytes and monocytic cell lines. Overexpression and depletion experiments were performed to assess CFIm25’s role in differentiation, NF-κB signaling, and alternative polyadenylation (APA). mRNA 3’ end-focused sequencing was conducted to identify changes in poly(A) site use of genes involved in macrophage differentiation and function. Cell cycle markers, NF-κB pathway components, and their targets were examined. The role of CFIm25 in NF-κB signaling was further evaluated through chemical inhibition and knockdown of pathway regulators.ResultsCFIm25 showed a striking increase upon macrophage differentiation, suggesting it promotes this process. Indeed, CFIm25 overexpression during differentiation amplified the acquisition of macrophage characteristics and caused an earlier slowing of the cell cycle, a hallmark of this transition, along with APA-mediated downregulation of cyclin D1. The NF-κB signaling pathway plays a major role in maturation of monocytes to macrophages, and the mRNAs of null, TBL1XR1, and NFKB1, all positive regulators of NF-κB signaling, underwent 3’UTR shortening, coupled with an increase in the corresponding proteins. CFIm25 overexpression also elevated phosphorylation of the NF-κB-p65 transcription activator, produced an earlier increase in the NF-κB targets p21, Bcl-XL, ICAM1 and TNF-α, and resulted in greater resistance to NF-κB chemical inhibition. Knockdown of Tables 2 and TBL1XR1 in CFIm25-overexpressing cells attenuated these effects, reinforcing the mechanistic link between CFIm25-regulated APA and NF-κB activation. Conversely, depletion of CFIm25 hindered differentiation and led to lengthening of NFKB1, TAB2, and TBL1XR1 3’ UTRs.ConclusionsOur study establishes CFIm25 as a key mediator of macrophage differentiation that operates through a coordinated control of cell cycle progression and NF-κB signaling. This linkage of mRNA processing and immune cell function also expands our understanding of the role of alternative polyadenylation in regulating cell signaling.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12964-025-02114-1.

ABSTRACTCell therapy based on chimeric antigen receptor (CAR) T cells has represented a revolutionary new approach for treating tumors, especially hematological diseases. Complete remission rates (CRR) > 80%–97% and 50%–90% overall response rates (ORR) have been achieved with a treatment based on CAR‐T cells in patients with malignant B‐cell tumors that have relapsed or are refractory to previous treatments. Toxicity remains the major problem. Most patients treated with CAR‐T cells develop high‐grade cytokine release syndrome (CRS) and immune effector cell‐associated neurotoxicity syndrome (ICANS). However, the unprecedentedly high CRR and ORR have led to the approval of six CAR‐T cell therapeutics by the Food and Drug Administration (FDA) and the European Medicines Agency (EMA), prompting researchers to improve existing products and develop new ones. By now, around 1000 clinical trials based on CAR‐T cells are registered at ClinicalTrials.gov: 82% are for hematological diseases, while the remaining 16% are for solid tumors. As a result of this increased research, an enormous amount of conflicting information has been accumulated in the literature, and each group follows its manufacturing protocols and performs specific in vitro testing. This review aimed to combine and compare clinical and preclinical information, highlighting the most used protocols to provide a comprehensive overview of the in vitro world of CAR‐T cells, from manufacturing to their characterization. The focus is on all steps of the CAR‐T cell manufacturing process, from the collection of patient or donor blood to the enrichment of T cells, their activation with anti‐CD3/CD28 beads, interleukin‐2 (IL‐2) or IL‐7 and IL‐15 (induction of a more functional memory phenotype), and their transfection (viral or non‐viral methods). Automation is crucial for ensuring a standardized final product.

Co-stimulatory molecules are imperative for CD8+ T cells to eliminate target cell and maintain sustained cytotoxicity. Despite an advanced understanding of the co-stimulatory molecules deficiency that results in tumor escape, the tumor cell-intrinsic mechanisms that regulate co-stimulatory molecules remain enigmatic, and an in-depth dissection could facilitate the improvement of treatment options. To this end, in this study, we report that the deficiency of the critical costimulatory molecule CD58, mediated by the expression of ATF4 in tumor cells, impairs the formation of immunological synapses (IS) and leads to the deterioration of antitumor immune function of CD8+ T cells. Mechanistically, ATF4 transcriptionally upregulated dynamin 1 (DNM1) expression leading to DNM1-dependent endocytosis (DDE)-mediated degradation of CD58. Furthermore, administration of DDE inhibitor prochlorperazine or ATF4 knockdown effectively restored CD58 expression, boosting CD8+ T cell cytotoxicity and immunotherapy efficiency. Thus, our study reveals that ATF4 in tumor cells weakens CD58 expression to interfere with complete IS formation, and indicates potential approaches to improve the cytolytic function of CD8+ T cell in tumor immunotherapy.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12967-025-06245-4.

Background/Objectives: The SARS-CoV-2’s high mutations and replication rates contribute to its high infectivity and resistance to current vaccinations and treatments. The primary cause of resistance to most current treatments aligns within the coding regions for the spike S protein of SARS-CoV-2 that has mutated. As a potential novel immunotherapy, we generated a novel fusion protein composed of a soluble ACE2 (sACE2) linked to llama-derived anti-CD16 that targets different variants of spike proteins and enhances natural killer cells to target infected cells. Methods: Here, we generated a novel sACE2-AntiCD16VHH fusion protein using a Gly4Ser linker, synthesized and cloned into the pLVX-EF1alpha-IRES-Puro vector, and further expressed in ExpiCHO-S cells and purified using Ni+NTA chromatography. Results: The fusion protein significantly blocked SARS-CoV-2 alpha, beta, delta, gamma, and omicron S-proteins binding and activating angiotensin-converting enzyme receptor-2 (ACE2) on ACE2-expressing RAW-Blue macrophage cells and the secretion of several key inflammatory cytokines, G-CSF, MIP-1A, and MCP-1, implicated in the cytokine release storm (CRS). The sACE2-Anti-CD16VHH fusion protein also bridged NK cells to ACE2-expressing human lung carcinoma A549 cells and significantly activated NK-dependent cytotoxicity. Conclusions: The findings show that a VHH directed against CD16 could be an excellent candidate to be linked to soluble ACE2 to generate a bi-specific molecule (sACE2-AntiCD16VHH) suitable for bridging effector cells and infected target cells to inhibit SARS-CoV-2 variant spike proteins binding to the ACE2 receptor in the RAW-Blue cell line and pro-inflammatory cytokines and to activate natural killer cell cytotoxicity.

Disease progression and drug resistance in patients with chronic lymphocytic leukaemia (CLL) depend on signals from the tumour microenvironment in lymphoid sites. GRK2 inhibits the egress of normal B cells from lymphoid tissues by inducing the downregulation of the S1P-receptor 1 (S1PR1). In this study we investigated the role of GRK2 in the context of CLL using in vitro and in vivo murine models, and also primary samples from CLL patients. We found that pharmacological inhibition of GRK2 enhanced the migration of leukemic cells from CLL patients towards S1P and impaired the S1P-induced downregulation of S1PR1. Likewise, CRISPR/Cas9-mediated GRK2 deletion in a murine leukemic cell line derived from the Eµ-TCL1 mouse model of CLL also increased migratory capacity toward S1P in vitro. Furthermore, when injected into mice, GRK2-deficient murine leukemic cells exhibited an altered in vivo localization, with a higher presence in the blood and spleen compared to the bone marrow. Within the spleen, these cells displayed reduced localization to the follicles compared to control murine leukemic cells. Deletion of GRK2 on murine leukemic cells did not affect their in vitro proliferation, but notably, conferred a growth disadvantage in vivo. These findings underscore GRK2 as a critical regulator of the localization of CLL cells in vivo and suggest its potential as a therapeutic target to disrupt survival niches in CLL.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-91536-5.

Current clinical strategies for the delivery of pulmonary therapeutics to the lung are primarily targeted to the upper portions of the airways, such as treatment with nebulized instillation and inhalation. However, targeted delivery to the lower regions of the lung is necessary for the treatment of parenchymal lung injury and disease. Here, we show the development of an mRNA therapeutic for the lower lung in mice using one-component Ionizable Amphiphilic Janus Dendrimers as a delivery vehicle. We deliver an anti-inflammatory cytokine mRNA, transforming growth factor-beta, to produce transient protein expression in the lower regions of the lung. This study highlights a method for precise, effective, and safe delivery of TGF-β mRNA to the lung in mice. This delivery system offers a promising approach for targeting therapeutics to the specific tissues, a strategy necessary to fill the current clinical gap in treating parenchymal lung injury and disease. Targeted delivery to the lower regions of the lung is necessary for the treatment of parenchymal lung injury and disease but is challenging. Here, the authors develop an mRNA delivery platform to treat acute lung injury in mice and demonstrate that it can reach the lower regions of the lung.