InVivoMAb polyclonal Armenian hamster IgG

Background: Immune checkpoint inhibitors are ineffective in the majority of colorectal cancers (CRCs) that are microsatellite stable. However, the underlying reasons for their unresponsiveness and mechanisms of immune evasion are poorly understood. In the present study, we aimed to elucidate the mechanisms underlying the immune evasion driven by CRC cells. Methods: We performed single-cell RNA sequencing of tumor tissues from 30 CRC patients and syngeneic mice implanted with transformation-related protein 53 (Trp53) knockout CT26 cells. Gene expression and correlations of individual tumor microenvironment (TME) components were analyzed, and their functional significance was investigated using syngeneic mouse models and cell line co-culture experiments. Results: CCAAT enhancer-binding protein beta (CEBPB) expression was increased in tumor protein 53 (TP53)-mutated CRCs. We confirmed that wild-type TP53 negatively regulated CEBPB expression in CRC cell lines. CEBPB expression was associated with decreased intratumoral T cell infiltration and negatively impacted survival in CRC patients. In the intercellular correlation analysis of gene expression, tumor epithelial cell CEBPB expression was significantly correlated with cytotoxic T-lymphocyte associated protein 4 (CTLA4) expression in T cells, especially in regulatory and exhausted T cells. Cebpb overexpression promoted tumor growth in the immunocompetent syngeneic mouse models, which was accompanied by increased CTLA-4 expression in tumor-infiltrating CD4+ T cells. In vitro co-culture experiments also showed that tumor cell CEBPB overexpression increased CTLA4 in T cells. Conclusions: Tumor cell CEBPB expression, up-regulated by TP53 mutation, can increase CTLA4 expression in T cells and negatively affect patient outcomes. These findings suggested a central role of tumor cell CEBPB in shaping an immunosuppressive TME.

Discovering more targets is of great importance for developing alternative interventions for tumor therapy. The roles of transmembrane protein 175 (TMEM175) in neurodegeneration diseases have been reported, however its functions in tumor immune surveillance are not known. We show that TMEM175 conditional knockout in macrophages inhibits the tumor growth and metastasis through promoting the anti-tumor immunity in the tumor microenvironment (TME), including elevated M1-like polarization, reduced M2-like polarization, and facilitated recruitment and activation of T cells and nature killer cells (NKs). The anti-tumor immunity is abrogated by caspase-1 inhibitor VX-765, anti-IL-1β, and anti-IL-18. Tmem175-/- bone marrow-derived macrophages (BMDMs) show enhanced tumor antigen cross-presentation that is further strengthened by IL-1β and IL-18. NLRP3 is robustly elicited in Tmem175-/- BMDMs by the tumor cell debris through lysosomal permeabilization and cathepsin B leakage. Finally, Tmem175-/- mice are more responsive to anti-PD-1. Our works implies TMEM175 to be a potential target for immunotherapy.

Dysfunctional white adipose tissue contributes to the development of obesity-related morbidities, including insulin resistance, dyslipidemia, and other metabolic disorders. Adipose tissue macrophages (ATMs) accumulate in obesity and play both beneficial and harmful roles in the maintenance of adipose tissue homeostasis and function. Despite their importance, the molecules and mechanisms that regulate these diverse functions are not well understood. Lipid-associated macrophages (LAMs), the dominant subset of obesity-associated ATMs, accumulate in crown-like structures and are characterized by a metabolically activated and proinflammatory phenotype. We previously identified CD9 as a surface marker of LAMs. However, the contribution of CD9 to the activation and function of LAMs during obesity is unknown. Using a myeloid-specific CD9 knockout model, we show that CD9 supports ATM-adipocyte adhesion and crown-like structure formation. Furthermore, CD9 promotes the expression of pro-fibrotic and extracellular matrix remodeling genes. Loss of myeloid CD9 reduces adipose tissue fibrosis, increases visceral adipose tissue accumulation, and improves global metabolic outcomes during diet-induced obesity. These results identify CD9 as a causal regulator of pathogenic LAM functions, highlighting CD9 as a potential therapeutic target for treating obesity-associated metabolic disease.

Peritonitis is an inflammation of the peritoneum primarily caused by gut perforation and consequent bacterial leakage, a known cause of sepsis. Although adipose tissue is recognized as an immunologically active organ, the involvement of adipose tissue innate lymphoid cells (ILC) in regulating peritonitis remains poorly understood. Here, we employ a cecal ligation and puncture mouse model and demonstrate that circulating CD127- group 1 ILC (ILC1) migrate into the mesenteric adipose tissue (MAT) during the inflammatory period of peritonitis. CD127- ILC1s undergo phenotypic changes to become CD127+ ILC1s, resulting in an increased number of CD127+ ILC1s in the MAT. We also show that this population of CD127+ ILC1s expresses PD-L1, exhibits low IFN-γ production, and potentially acts as a negative regulator of TNF production by γδ T cells, thereby controlling acute peritonitis. Our findings suggest that MAT-CD127+ ILC1s play an important regulatory role in acute peritonitis and may represent a potential therapeutic target for sepsis.

In Juvenile Idiopathic Arthritis (JIA), the most common childhood rheumatic disease, many patients also develop uveitis (JIA-uveitis), risking life-long vision loss. The mechanisms driving uveitis development in JIA remain understudied. Here, we demonstrate that peripheral blood CD19+IgD-CD27- double negative type 1 (DN1) B cells are elevated in JIA-uveitis compared to JIA patients without eye disease (JIA). The B cell receptor (BCR) repertoire was also more clonal and somatically hypermutated in JIA-uveitis and antigen-activated B cells infiltrated chronically inflamed JIA-uveitis eyes. Features of heightened B cell activation were recapitulated in experimental autoimmune uveoretinitis (EAU) and disrupting B and T cell interactions using monoclonal antibodies and transgenic mice suppresses uveitis. Together, these findings support a conceptual shift that uveitis is a primarily T cell driven disease and provide evidence for potential new therapeutic strategies that also consider B cells as drivers in disease pathology.

Background: Lung metastasis is a leading cause of breast cancer (BC)-related mortality, driven by the immunosuppressive traits of the metastatic tumor microenvironment. However, the mechanisms underlying cell-cell crosstalk in shaping immune evasion within the metastatic niche remain poorly defined. Neutrophil extracellular traps (NETs) and their associated proteins, such as cathelicidin, have emerged as key mediators of metastatic regulation in cancer. Here, we aimed to decipher the interaction between a neutrophil subset characterized by high expression of lymphocyte antigen 6 complex locus g (Ly6ghigh) and cluster of differentiation 8-positive T lymphocytes (CD8+ T cells), mediated via cathelicidin embedded in NETs, as well as their synergistic mechanism and cooperative role in promoting lung metastasis of BC. Methods: We characterized neutrophil heterogeneity and functional dynamics by performing single-cell RNA sequencing and flow cytometry on lung tissues derived from murine models of BC lung metastasis. We utilized cathelicidin-related antimicrobial peptide (Cramp) knockout mice to dissect the role of cathelicidin in NETs. The spatial colocalization of apoptotic CD8+ T cells and NETs was analyzed using multiplex immunofluorescence, and the molecular interactions were probed by protein binding assays. Results: Neutrophils in the lung metastatic niche were classified into 2 subsets based on the Ly6g expression: Ly6ghigh and Ly6glow neutrophils. Ly6glow neutrophils, which were recruited in the macrometastatic stage, exhibited myeloid-derived suppressor cell-like characteristics. Notably, Ly6ghigh neutrophils induced CD8+ T cell apoptosis through NET formation, with apoptotic CD8+ T cells spatially clustered within NET-rich areas. Mechanistically, NET-derived cathelicidin (Cramp in mice) directly bound to mitochondrial adenine nucleotide translocator 1 (Ant1) in CD8+ T cells, triggering conformational changes and complex formation with voltage-dependent anion channel 1 (Vdac1). These events resulted in the opening of the mitochondrial permeability transition pore and loss of mitochondrial membrane potential. Conclusions: Our study demonstrates that Ly6ghigh neutrophils play a critical role in immunosuppression and immune evasion through NET-induced apoptosis of CD8+ T cells. These findings underscore the importance of NETs and cathelicidin in BC lung metastasis, suggesting their potential as therapeutic targets in restoring antitumor immunity and in preventing metastatic progression.

Dendritic cells are essential for establishing thymic central tolerance; however, mechanisms supporting their homeostasis and activation remain unresolved. Through single-cell transcriptomics and functional assays, we identify seven thymic conventional dendritic cell (cDC) subsets and discriminate their abilities to present self-antigens and induce regulatory T cells. Mice blocked at different stages of T cell development revealed that CD4+ single-positive (CD4SP) and CD8SP thymocytes differentially support homeostasis and activation of type 1 cDCs (cDC1s) versus cDC2s/plasmacytoid DCs (pDCs), respectively. CD8SP thymocytes indirectly support pDC survival and cDC2 thymic migration, and they induce interferon signaling in cDCs, partly by promoting type 3 interferon expression by medullary thymic epithelial cells. By contrast, CD4SP thymocytes undergo cognate interactions with cDCs, inducing CD40 signaling required for activation of cDC1s. Activated cDC1s make nonredundant contributions to central tolerance. Together, this study comprehensively identifies distinct thymic DC subsets and elucidates requirements for cross-talk with thymocyte subsets that support their homeostasis, activation and function.

An adaptive immune response to cancer requires three main signals: antigen presentation and recognition ("signal 1"), costimulation ("signal 2"), and secreted immunostimulatory cytokines ("signal 3"). Expression of these signals in tumors via non-viral gene delivery represents a promising strategy to reprogram the tumor microenvironment (TME) and prime antitumor immunity.

Modulating harmful neuroimmune responses is a promising therapeutic approach for hemorrhagic stroke, a condition that still lacks effective treatment. The immune checkpoint B and T Lymphocyte Attenuator (BTLA) helps suppress immune activation; however, its role in intracerebral hemorrhage (ICH) remains unclear. This study explores whether a BTLA-activating antibody can reduce neuroinflammation, mitigate brain injury, improve recovery after ICH, and elucidate the underlying mechanisms.

Rising obesity rates are closely linked to higher risk of cancer, yet the underlying mechanisms are not fully understood. It is previously reported that fatty acids (FAs) released from cancer-associated adipose tissue enhance hypoxia-inducible factor-1α (HIF-1α) expression in cancer cells, promoting tumor progression. Here, it is elucidated that cancer cells manipulate adjacent adipose tissue by secreting C-C chemokine ligand2 (CCL2) to exploit FAs. Activation of HIF-1α induced by FA influx increases CCL2 expression in cancer cells, which subsequently leads to lipolysis in nearby adipose tissue by activating peroxisome proliferator-activated receptor alpha (PPARα) signaling. This activation in adipose tissue results in the release of FAs into the tumor microenvironment. The increased lipid supply to tumor reactivates the FA/HIF-1α/CCL2 axis in cancer cells, further accelerating tumor growth and CCL2 secretion. This establishes a positive feedback loop between tumor and adjacent adipose tissue, which enhances cancer progression. This crosstalk is validated by using a polydimethylsiloxane-based 3D coculture system and in vivo models. In obese mice, this reciprocal signaling accelerated tumor progression, whereas intra-tumoral injection of CCL2-neutralizing antibody significantly suppressed it. These findings reveal a metabolic circuit for tumor survival and disrupting this interaction may provide promising therapeutic targets, particularly for obese cancer patients.

CD8+ virtual memory T (TVM) cells rapidly respond to infection via antigen-independent bystander effector functions. While it is recognized that TVM cells arise independently of foreign antigen encounter, the mechanisms governing their development are not fully understood. Here, we identify the Ikaros transcription factor Aiolos as a negative regulator of TVM cell programming. We observe enhanced frequencies and numbers of TVM in the spleen, liver, and blood of unchallenged Aiolos-deficient (Ikzf3-/-) mice and in the lungs 1-day post-infection with influenza A virus (IAV). Furthermore, Ikzf3-/- TVM cells produce elevated IFN-γ and granzyme B in response to cytokine stimulation. Importantly, Aiolos-deficient mice control IAV more rapidly and exhibit reduced morbidity, indicating enhanced TVM cell functionality. Mechanistically, Aiolos represses the expression of the transcription factor Eomes and the IL-15R subunit CD122, known positive regulators of TVM gene program. Collectively, these findings establish Aiolos as a molecular repressor of TVM programming and responses.

Ki-ras2 (KRAS) mutation is a common driver of lung cancer, and KRAS-mutated tumors are relatively resistant to radiotherapy. Previously, we demonstrated that mitogen-activated protein kinase (MEK) inhibitors (MEKi) enhanced treatment efficacy by increasing the anti-tumor immune response after radiotherapy in KRAS-mutant tumors. In this study, we explored the potential mechanism underlying the MEKi-mediated increase in anti-tumor immune response.

Liposarcoma is the most common soft tissue sarcoma. Well-differentiated liposarcoma can progress to dedifferentiated liposarcoma (DDLPS), a more aggressive form with higher metastatic potential and poor response to existing therapies. Progress in understanding and treating liposarcoma has been limited. To address this, we sought to develop an immunocompetent genetically engineered mouse model of liposarcoma.

In the non-obese diabetic (NOD) mouse model of autoimmune diabetes, interleukin (IL)-27 stimulates interferon γ (IFNγ) production by CD4 and CD8 T cells and is essential for disease development. Here, we tested the role of IL-27 in cellular communication. Single-cell RNA sequencing and T cell adoptive transfer showed that IL-27 intrinsically controlled the differentiation of islet-infiltrating CD4 T cells by driving them toward an IL-21+ Th1 phenotype. Consequently, IL-27 signaling in CD4 T cells was important for BATF and granzyme B expression in islet CD8 T effectors. BATF overexpression increased the diabetogenic potential of β cell autoreactive CD8 T cells lacking help from CD4 T cell-derived IL-21. Macrophages were the main source of IL-27 in the islets, whose expression correlated with T cell infiltration. IFNγ and CD40 signaling conferred by activated T cells induced macrophage IL-27 production. Collectively, our findings reveal a role for IL-27 in orchestrating interconnected positive feedback loops involving CD4 T cells, CD8 T cells, and macrophages in autoimmune diabetes.

Psoriasis is a chronic, complex immune-mediated inflammatory disorder with cutaneous and systemic manifestations in which keratinocytes, dendritic cells and T cells have central roles. UBE2L3 may be a protective biomarker that regulates the pathogenesis of psoriasis. Here, we identify the IL-17A signaling similarity between human psoriatic skin and Ube2l3 conditional knockout mouse skin in the epidermis rather than dermis. IL-17A is regulated by CXCR6+ Vγ2+ γδT cells in mouse while CXCR6+ CD8+ T cells in human. CXCL16 is the only chemokine that binds to and stimulates CXCR6. Ube2l3 reduction in keratinocytes activates IL-1β and then promotes CXCL16 expression through STAT3 signaling. Up-regulated CXCL16 in keratinocytes and cDC2/mDC then attracts Vγ2+ γδT17 or CD8+ T cells to secrete IL-17A and form a positive feedback loop in keratinocytes supporting psoriatic lesions. Thus, UBE2L3 is a keratinocyte-intrinsic suppressor of epidermal IL-17 production in Vγ2+ γδT cells in mouse and CD8+ T cells in human through the CXCL16/CXCR6 signaling pathway in psoriasis.

Systemic lupus erythematosus (SLE) is an autoimmune disease driven by autoantibody production. Lupus nephritis (LN), a severe SLE complication, is primarily caused by renal autoantibodies. Long-lived plasma cells (LLPCs), the main producers of these autoantibodies, are especially elevated in the kidney of LN patients, particularly in refractory or recurrent cases. However, the cause of increased LLPCs in LN kidneys remains unknown. This study uses an LN mouse model and combines single-cell RNA sequencing with spatial transcriptomics, finding that kidney-resident Cxcl9high macrophages and their secreted chemokine Cxcl9 significantly rise with disease progression. This increase in Cxcl9 attracts Cxcr3+ plasmablasts in peripheral blood into the kidneys, where they differentiate into LLPCs and produce autoantibodies. Based on these findings, this study suggests that Cxcl9high macrophages are the inducing factor causing the increase of LLPCs in LN kidneys and may be a potential therapeutic target for LN.

Inflammatory bowel disease (IBD) is characterized by chronic, relapsing inflammation of the gastrointestinal tract. Genetic factors, including variants in T-cell activation Rho GTPase-activating protein (TAGAP), contribute to disease susceptibility and severity.

Intraepithelial lymphocytes expressing the γδ T cell receptor (γδ IEL) provide continuous surveillance of the intestinal epithelium. We report that mice harboring a microbiota-specific hyperproliferative γδ IEL (γδHYP) phenotype also upregulate the expression of the ectonucleotidase CD39, a marker of regulatory γδ T cells. Enhanced TCR and IL-15 signaling correlates with a progression from a naïve-like CD39neg γδ IEL to a more mature, tissue-adapted CD39hi IEL population. We found that TCRγδ activation drives CD122-mediated CD39 upregulation on γδHYP IELs and increased mucosal IL-15 further amplifies CD39 expression in these cells. Further investigation revealed that CD39 induction requires sustained exposure to the γδHYP-associated microbiota. Moreover, CD39hi γδ IELs exhibit a reduced capacity to produce pro-inflammatory cytokine, which may explain the lack of histopathology in γδHYP mice. Overall, our study identifies a previously unappreciated mechanism by which an altered microbiota amplifies CD39 expression on γδHYP IELs, leading to the expansion of γδ IELs with regulatory potential.

Tumour necrosis is associated with poor prognosis in cancer1,2 and is thought to occur passively when tumour growth outpaces nutrient supply. Here we report, however, that neutrophils actively induce tumour necrosis. In multiple cancer mouse models, we found a tumour-elicited Ly6GHighLy6CLow neutrophil population that was unable to extravasate in response to inflammatory challenges but formed neutrophil extracellular traps (NETs) more efficiently than classical Ly6GHighLy6CHigh neutrophils. The presence of these 'vascular-restricted' neutrophils correlated with the appearance of a 'pleomorphic' necrotic architecture in mice. In tumours with pleomorphic necrosis, we found intravascular aggregates of neutrophils and NETs that caused occlusion of the tumour vasculature, driving hypoxia and necrosis of downstream vascular beds. Furthermore, we found that cancer cells adjacent to these necrotic regions (that is, in 'perinecrotic' areas) underwent epithelial-to-mesenchymal transition, explaining the paradoxical metastasis-enhancing effect of tumour necrosis. Blocking NET formation genetically or pharmacologically reduced the extent of tumour necrosis and lung metastasis. Thus, by showing that NETs drive vascular occlusion, pleomorphic necrosis and metastasis, we demonstrate that tumour necrosis is not necessarily a passive byproduct of tumour growth and that it can be blocked to reduce metastatic spread.

Dendritic cells (DCs) are key regulators of adaptive immunity, guiding T helper (Th) cell differentiation through antigen presentation, co-stimulation, and cytokine production. However, in steady-state conditions, certain DC subsets, such as Langerhans cells (LCs), induce T follicular helper (Tfh) cells and B cell responses without inflammatory stimuli. Using multiple mouse models and in vitro systems, we investigated the mechanisms underlying steady-state LC-induced adaptive immune responses. We found that LCs drive germinal center Tfh and B cell differentiation and antibody production independently of interleukin-6 (IL-6), type-I interferons, and ICOS ligand (ICOS-L) signaling, which are critical in inflammatory settings. Instead, these responses relied on CD80/CD86-mediated co-stimulation. Our findings challenge the conventional three-signal paradigm by demonstrating that canonical cytokine signaling is dispensable for LC-mediated Tfh and B cell responses in steady-state. These insights provide a framework for understanding homeostatic immunity and the immune system's role in maintaining tolerance or developing autoimmunity under non-inflammatory conditions.