InVivoMAb anti-mouse CD16/CD32
Product Description
Specifications
| Isotype | Rat IgG2b, κ |
|---|---|
| Recommended Isotype Control(s) | InVivoMAb rat IgG2b isotype control, anti-keyhole limpet hemocyanin |
| Recommended Dilution Buffer | InVivoPure pH 8.0 Dilution Buffer |
| Conjugation | This product is unconjugated. Conjugation is available via our Antibody Conjugation Services. |
| Immunogen | BALB/c mouse macrophage cell line J774 |
| Reported Applications |
in vivo Fc receptor blocking Fc receptor blocking, flow cytometry Fc receptor blocking, immunofluorescence |
| Formulation |
PBS, pH 8.0 Contains no stabilizers or preservatives |
| Endotoxin |
≤1EU/mg (≤0.001EU/μg) Determined by LAL gel clotting assay |
| Purity |
≥95% Determined by SDS-PAGE |
| Sterility | 0.2 µm filtration |
| Production | Purified from cell culture supernatant in an animal-free facility |
| Purification | Protein G |
| RRID | AB_2736987 |
| Molecular Weight | 150 kDa |
| Storage | The antibody solution should be stored at the stock concentration at 4°C. Do not freeze. |
| Need a Custom Formulation? | See All Antibody Customization Options |
Application References
in vivo blocking of CD40/CD40L signaling
in vitro Fc receptor blocking
Pasqual, G., et al. (2018). "Monitoring T cell-dendritic cell interactions in vivo by intercellular enzymatic labelling" Nature 553(7689): 496-500.
PubMed
Interactions between different cell types are essential for multiple biological processes, including immunity, embryonic development and neuronal signalling. Although the dynamics of cell-cell interactions can be monitored in vivo by intravital microscopy, this approach does not provide any information on the receptors and ligands involved or enable the isolation of interacting cells for downstream analysis. Here we describe a complementary approach that uses bacterial sortase A-mediated cell labelling across synapses of immune cells to identify receptor-ligand interactions between cells in living mice, by generating a signal that can subsequently be detected ex vivo by flow cytometry. We call this approach for the labelling of ‘kiss-and-run’ interactions between immune cells ‘Labelling Immune Partnerships by SorTagging Intercellular Contacts’ (LIPSTIC). Using LIPSTIC, we show that interactions between dendritic cells and CD4(+) T cells during T-cell priming in vivo occur in two distinct modalities: an early, cognate stage, during which CD40-CD40L interactions occur specifically between T cells and antigen-loaded dendritic cells; and a later, non-cognate stage during which these interactions no longer require prior engagement of the T-cell receptor. Therefore, LIPSTIC enables the direct measurement of dynamic cell-cell interactions both in vitro and in vivo. Given its flexibility for use with different receptor-ligand pairs and a range of detectable labels, we expect that this approach will be of use to any field of biology requiring quantification of intercellular communication.
in vivo Fc receptor blocking
Arlauckas SP, Garris CS, Kohler RH, Kitaoka M, Cuccarese MF, Yang KS, Miller MA, Carlson JC, Freeman GJ, Anthony RM, Weissleder R, Pittet MJ. (2017). "In vivo imaging reveals a tumor-associated macrophage-mediated resistance pathway in anti-PD-1 therapy" Sci Transl Med 9(389):eaal3604.
PubMed
Monoclonal antibodies (mAbs) targeting the immune checkpoint anti-programmed cell death protein 1 (aPD-1) have demonstrated impressive benefits for the treatment of some cancers; however, these drugs are not always effective, and we still have a limited understanding of the mechanisms that contribute to their efficacy or lack thereof. We used in vivo imaging to uncover the fate and activity of aPD-1 mAbs in real time and at subcellular resolution in mice. We show that aPD-1 mAbs effectively bind PD-1+ tumor-infiltrating CD8+ T cells at early time points after administration. However, this engagement is transient, and aPD-1 mAbs are captured within minutes from the T cell surface by PD-1- tumor-associated macrophages. We further show that macrophage accrual of aPD-1 mAbs depends both on the drug's Fc domain glycan and on Fcγ receptors (FcγRs) expressed by host myeloid cells and extend these findings to the human setting. Finally, we demonstrate that in vivo blockade of FcγRs before aPD-1 mAb administration substantially prolongs aPD-1 mAb binding to tumor-infiltrating CD8+ T cells and enhances immunotherapy-induced tumor regression in mice. These investigations yield insight into aPD-1 target engagement in vivo and identify specific Fc/FcγR interactions that can be modulated to improve checkpoint blockade therapy.
in vitro Fc receptor blocking
in vivo IL-7 receptor stimulation (as a complex with IL-7)
Arbelaez, C. A., et al. (2015). "IL-7/IL-7 Receptor Signaling Differentially Affects Effector CD4+ T Cell Subsets Involved in Experimental Autoimmune Encephalomyelitis" J Immunol 195(5): 1974-1983.
PubMed
IL-17-producing CD4(+) T (Th17) cells, along with IFN-gamma-expressing Th1 cells, represent two major pathogenic T cell subsets in experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis (MS). The cytokines and transcription factors involved in the development and effector functions of Th1 and Th17 cells have been largely characterized. Among them, IL-23 is essential for the generation of stable and encephalitogenic Th17 cells and for the development of EAE. The IL-7/IL-7R signaling axis participates in cell survival, and perturbation of this pathway has been associated with enhanced susceptibility to MS. A link between IL-23-driven pathogenic T cells and IL-7/IL-7R signaling has previously been proposed, but has not been formally addressed. In the current study, we showed that Th17 cells from mice with EAE express high levels of IL-7Ralpha compared with Th1 cells. Using mice that constitutively express IL-7Ralpha on T cells, we determined that sustained IL-7R expression in IL-23R-deficient mice could not drive pathogenic T cells and the development of EAE. IL-7 inhibited the differentiation of Th17 cells, but promoted IFN-gamma and GM-CSF secretion in vitro. In vivo IL-7/anti-IL-7 mAb complexes selectively expanded and enhanced the proliferation of CXCR3-expressing Th1 cells, but did not impact Th17 cells and EAE development in wild-type and IL-23R-deficient mice. Importantly, high IL-7 expression was detected in the CNS during EAE and could drive the plasticity of Th17 cells to IFN-gamma-producing T cells. Together, these data address the contribution of IL-23/IL-23R and IL-7/IL-7R signaling in Th17 and Th1 cell dynamics during CNS autoimmunity.
in vivo LFA-1 neutralization
Immunofluorescence
in vivo MAdCAM-1 neutralization
in vitro Fc receptor blocking
Peske, J. D., et al. (2015). "Effector lymphocyte-induced lymph node-like vasculature enables naive T-cell entry into tumours and enhanced anti-tumour immunity" Nat Commun 6: 7114.
PubMed
The presence of lymph node (LN)-like vasculature in tumours, characterized by expression of peripheral node addressin and chemokine CCL21, is correlated with T-cell infiltration and positive prognosis in breast cancer and melanoma patients. However, mechanisms controlling the development of LN-like vasculature and how it might contribute to a beneficial outcome for cancer patients are unknown. Here we demonstrate that LN-like vasculature is present in murine models of melanoma and lung carcinoma. It enables infiltration by naive T cells that significantly delay tumour outgrowth after intratumoral activation. Development of this vasculature is controlled by a mechanism involving effector CD8 T cells and NK cells that secrete LTalpha3 and IFNgamma. LN-like vasculature is also associated with organized aggregates of B lymphocytes and gp38(+) fibroblasts, which resemble tertiary lymphoid organs that develop in models of chronic inflammation. These results establish LN-like vasculature as both a consequence of and key contributor to anti-tumour immunity.
in vivo CD47 blockade
in vivo IFNAR-1 blockade
in vitro Fc receptor blocking
in vivo CSF1 neutralization
Liu, X., et al. (2015). "CD47 blockade triggers T cell-mediated destruction of immunogenic tumors" Nat Med 21(10): 1209-1215.
PubMed
Macrophage phagocytosis of tumor cells mediated by CD47-specific blocking antibodies has been proposed to be the major effector mechanism in xenograft models. Here, using syngeneic immunocompetent mouse tumor models, we reveal that the therapeutic effects of CD47 blockade depend on dendritic cell but not macrophage cross-priming of T cell responses. The therapeutic effects of anti-CD47 antibody therapy were abrogated in T cell-deficient mice. In addition, the antitumor effects of CD47 blockade required expression of the cytosolic DNA sensor STING, but neither MyD88 nor TRIF, in CD11c(+) cells, suggesting that cytosolic sensing of DNA from tumor cells is enhanced by anti-CD47 treatment, further bridging the innate and adaptive responses. Notably, the timing of administration of standard chemotherapy markedly impacted the induction of antitumor T cell responses by CD47 blockade. Together, our findings indicate that CD47 blockade drives T cell-mediated elimination of immunogenic tumors.
Flow Cytometry
in vivo Fc receptor blocking
in vivo administration
Yu, X., et al. (2015). "A monoclonal antibody with anti-D-like activity in murine immune thrombocytopenia requires Fc domain function for immune thrombocytopenia ameliorative effects" Transfusion 55(6 Pt 2): 1501-1511.
PubMed
BACKGROUND: The mechanism of action of anti-D in ameliorating immune thrombocytopenia (ITP) remains unclear. The monoclonal antibody (MoAb) Ter119, which targets murine red blood cells (RBCs), has been shown to mimic the effect of anti-D in improving antibody-mediated murine ITP. The mechanism of Ter119-mediated ITP amelioration, especially the role of the antigen-binding and Fc domains, remains untested. A functional Fc domain is crucial for many therapeutic MoAb activity; therefore, the requirement of Ter119 Fc domain in ITP amelioration is investigated using outbred CD-1 mice. STUDY DESIGN AND METHODS: Ter119 variants, including Ter119 F(ab’)2 fragments, deglycosylated Ter119, and afucosylated Ter119, were generated to test their effect in ameliorating antibody-induced murine ITP. In vivo inhibition of FcgammaRIII and FcgammaRIIB was achieved using the Fab fragment of the FcgammaRIII/FcgammaRIIB-specific MoAb 2.4G2. RESULTS: Ter119 F(ab’)2 fragments and deglycosylated Ter119 were unable to ameliorate murine ITP or mediate phagocytosis of RBCs by RAW264.7 macrophages in vitro. Inhibition of FcgammaRIII and FcgammaRIIB, as well as Ter119 defucosylation, do not affect Ter119-mediated ITP amelioration. CONCLUSION: The Fc domain of Ter119, as well as its Fc glycosylation, is required for Ter119-mediated ITP amelioration. Moreover, both Fc and Fc glycosylation are required for Ter119-mediated phagocytosis in vitro. These findings demonstrate the importance of the Fc domain in a therapeutic MoAb with anti-D-like activity.
in vitro Fc receptor blocking
Heesch, K., et al. (2014). "The function of the chemokine receptor CXCR6 in the T cell response of mice against Listeria monocytogenes" PLoS One 9(5): e97701.
PubMed
The chemokine receptor CXCR6 is expressed on different T cell subsets and up-regulated following T cell activation. CXCR6 has been implicated in the localization of cells to the liver due to the constitutive expression of its ligand CXCL16 on liver sinusoidal endothelial cells. Here, we analyzed the role of CXCR6 in CD8+ T cell responses to infection of mice with Listeria monocytogenes. CD8+ T cells responding to listerial antigens acquired high expression levels of CXCR6. However, deficiency of mice in CXCR6 did not impair control of the L. monocytogenes infection. CXCR6-deficient mice were able to generate listeria-specific CD4+ and CD8+ T cell responses and showed accumulation of T cells in the infected liver. In transfer assays, we detected reduced accumulation of listeria-specific CXCR6-deficient CD8+ T cells in the liver at early time points post infection. Though, CXCR6 was dispensable at later time points of the CD8+ T cell response. When transferred CD8+ T cells were followed for extended time periods, we observed a decline in CXCR6-deficient CD8+ T cells. The manifestation of this cell loss depended on the tissue analyzed. In conclusion, our results demonstrate that CXCR6 is not required for the formation of a T cell response to L. monocytogenes and for the accumulation of T cells in the infected liver but CXCR6 appears to influence long-term survival and tissue distribution of activated cells.
in vivo CD8+ T cell depletion
in vivo PD-L1 blockade
in vitro Fc receptor blocking
ELISPOT
in vivo TNFα neutralization
in vitro IFNγ neutralization
in vivo MDSC depletion
Deng, L., et al. (2014). "Irradiation and anti-PD-L1 treatment synergistically promote antitumor immunity in mice" J Clin Invest 124(2): 687-695.
PubMed
High-dose ionizing irradiation (IR) results in direct tumor cell death and augments tumor-specific immunity, which enhances tumor control both locally and distantly. Unfortunately, local relapses often occur following IR treatment, indicating that IR-induced responses are inadequate to maintain antitumor immunity. Therapeutic blockade of the T cell negative regulator programmed death-ligand 1 (PD-L1, also called B7-H1) can enhance T cell effector function when PD-L1 is expressed in chronically inflamed tissues and tumors. Here, we demonstrate that PD-L1 was upregulated in the tumor microenvironment after IR. Administration of anti-PD-L1 enhanced the efficacy of IR through a cytotoxic T cell-dependent mechanism. Concomitant with IR-mediated tumor regression, we observed that IR and anti-PD-L1 synergistically reduced the local accumulation of tumor-infiltrating myeloid-derived suppressor cells (MDSCs), which suppress T cells and alter the tumor immune microenvironment. Furthermore, activation of cytotoxic T cells with combination therapy mediated the reduction of MDSCs in tumors through the cytotoxic actions of TNF. Our data provide evidence for a close interaction between IR, T cells, and the PD-L1/PD-1 axis and establish a basis for the rational design of combination therapy with immune modulators and radiotherapy.
in vitro Fc receptor blocking
in vitro B cell stimulation/activation
Muppidi, J. R., et al. (2014). "Loss of signalling via Galpha13 in germinal centre B-cell-derived lymphoma" Nature 516(7530): 254-258.
PubMed
Germinal centre B-cell-like diffuse large B-cell lymphoma (GCB-DLBCL) is a common malignancy, yet the signalling pathways that are deregulated and the factors leading to its systemic dissemination are poorly defined. Work in mice showed that sphingosine-1-phosphate receptor-2 (S1PR2), a Galpha12 and Galpha13 coupled receptor, promotes growth regulation and local confinement of germinal centre B cells. Recent deep sequencing studies of GCB-DLBCL have revealed mutations in many genes in this cancer, including in GNA13 (encoding Galpha13) and S1PR2 (refs 5,6, 7). Here we show, using in vitro and in vivo assays, that GCB-DLBCL-associated mutations occurring in S1PR2 frequently disrupt the receptor’s Akt and migration inhibitory functions. Galpha13-deficient mouse germinal centre B cells and human GCB-DLBCL cells were unable to suppress pAkt and migration in response to S1P, and Galpha13-deficient mice developed germinal centre B-cell-derived lymphoma. Germinal centre B cells, unlike most lymphocytes, are tightly confined in lymphoid organs and do not recirculate. Remarkably, deficiency in Galpha13, but not S1PR2, led to germinal centre B-cell dissemination into lymph and blood. GCB-DLBCL cell lines frequently carried mutations in the Galpha13 effector ARHGEF1, and Arhgef1 deficiency also led to germinal centre B-cell dissemination. The incomplete phenocopy of Galpha13- and S1PR2 deficiency led us to discover that P2RY8, an orphan receptor that is mutated in GCB-DLBCL and another germinal centre B-cell-derived malignancy, Burkitt’s lymphoma, also represses germinal centre B-cell growth and promotes confinement via Galpha13. These findings identify a Galpha13-dependent pathway that exerts dual actions in suppressing growth and blocking dissemination of germinal centre B cells that is frequently disrupted in germinal centre B-cell-derived lymphoma.
in vitro Fc receptor blocking
in vivo TGFβ neutralization
Leon, B., et al. (2014). "FoxP3+ regulatory T cells promote influenza-specific Tfh responses by controlling IL-2 availability" Nat Commun 5: 3495.
PubMed
Here, we test the role of FoxP3(+) regulatory T cells (Tregs) in controlling T follicular helper (Tfh) and germinal centre (GC) B-cell responses to influenza. In contrast to the idea that Tregs suppress T-cell responses, we find that Treg depletion severely reduces the Tfh cell response to influenza virus. Furthermore, Treg depletion prevents the accumulation of influenza-specific GCs. These effects are not due to alterations in TGFbeta availability or a precursor-progeny relationship between Tregs and Tfh cells, but are instead mediated by increased availability of IL-2, which suppresses the differentiation of Tfh cells and as a consequence, compromises the GC B response. Thus, Tregs promote influenza-specific GC responses by preventing excessive IL-2 signalling, which suppresses Tfh cell differentiation.
Immunofluorescence
Fc receptor blocking
Brinkman CC, Rouhani SJ, Srinivasan N, Engelhard VH. (2013). "Peripheral tissue homing receptors enable T cell entry into lymph nodes and affect the anatomical distribution of memory cells" J Immunol 191(5):2412-25.
PubMed
Peripheral tissue homing receptors enable T cells to access inflamed nonlymphoid tissues. In this study, we show that two such molecules, E-selectin ligand and α4β1 integrin, enable activated and memory T cells to enter lymph nodes (LN) as well. This affects the quantitative and qualitative distribution of these cells among regional LN beds. CD8 memory T cells in LN that express these molecules were mostly CD62L(lo) and would normally be classified as effector memory cells. However, similar to central memory cells, they expanded upon Ag re-encounter. This led to differences in the magnitude of the recall response that depended on the route of immunization. These novel cells share properties of both central and effector memory cells and reside in LN based on previously undescribed mechanisms of entry.
Product Citations
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A non-genotoxic stem cell therapy boosts lymphopoiesis and averts age-related blood diseases in mice.
In Nat Commun on 2 June 2025 by Konturek-Ciesla, A., Zhang, Q., et al.
PubMed
Hematopoietic stem cell (HSC) transplantation offers a cure for a variety of blood disorders, predominantly affecting the elderly; however, its application, especially in this demographic, is limited by treatment toxicity. In response, we employ a murine transplantation model based on low-intensity conditioning protocols using antibody-mediated HSC depletion. While aging presents a significant barrier to effective HSC engraftment, optimizing HSC doses and non-genotoxic targeting methods greatly enhance the long-term multilineage activity of the transplanted cells. We demonstrate that young HSCs, once effectively engrafted in aged hosts, improve hematopoietic output and ameliorate age-compromised lymphopoiesis. This culminated in a strategy that robustly mitigates disease progression in a genetic model of myelodysplastic syndrome. These results suggest that non-genotoxic HSC transplantation could fundamentally change the clinical management of age-associated hematological disorders, offering a prophylactic tool to delay or even prevent their onset in elderly patients.
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WNK1 mediates M-CSF-induced macropinocytosis to enforce macrophage lineage fidelity.
In Nat Commun on 28 May 2025 by Trzeciak, A., Liu, Z. L., et al.
PubMed
Tissue-resident macrophages (TRM) are critical for mammalian organismal development and homeostasis. Here we report that with-no-lysine 1 (WNK1) controls myeloid progenitor fate, with Csf1riCre-mediated Wnk1 deletion in mice (WNK1-deficient mice) resulting in loss of TRMs and causing perinatal mortality. Mechanistically, absence of WNK1 or inhibition of WNK kinase activity disrupts macrophage colony-stimulating factor (M-CSF)-stimulated macropinocytosis, thereby blocking mouse and human progenitor and monocyte differentiation into macrophages and skewing progenitor differentiation into neutrophils. Treatment with PMA rescues macropinocytosis but not macrophage differentiation of WNK-inhibited progenitors, implicating that M-CSF-stimulated, macropinocytosis-induced activation of WNK1 is required for macrophage differentiation. Finally, M-CSF-stimulated macropinocytosis triggers WNK1 nuclear translocation and concomitant increased protein expression of interferon regulatory factor (IRF)8, whereas inhibition of macropinocytosis or WNK kinase activity suppresses IRF8 expression. Our results thus suggest that WNK1 and downstream IRF8-regulated genes are important for M-CSF/macropinocytosis-mediated regulation of myeloid cell lineage commitment during TRM development and homeostasis.
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Tracking Oral Nanoparticle Uptake in Mouse Gastrointestinal Tract by Fluorescent Labeling and t-SNE Flow Cytometry.
In Bio Protoc on 20 May 2025 by Mow, R. J., Kuczma, M. P., et al.
PubMed
The growing demand for advanced analytical techniques to explore complex cellular targets of nanotherapeutics has driven the development of innovative methodologies. This protocol presents a refined approach for fluorescent labeling and flow cytometric analysis of colonic cells following oral lipid nanoparticle (LNP) treatment, focusing on LNP uptake in colonic cell subpopulations in a DSS-induced colitis mouse model. By integrating optimized fluorochrome selection and gating strategies with advanced t-distributed stochastic neighbor embedding (t-SNE) analysis, this method enables precise identification and multidimensional visualization of LNP-targeted epithelial and macrophage populations under the complex conditions of inflamed colon tissue. Building on our previous studies demonstrating the effectiveness of nanoparticles in targeted drug delivery, this approach highlights the utility of flow cytometry for assessing uptake efficiency and cellular targeting. Unlike conventional protocols, it incorporates t-SNE for enhanced multidimensional analysis, allowing for the detection of subtle cellular patterns and the delineation of intricate clusters. By addressing gaps in traditional methodologies, this protocol provides a robust and reproducible framework for investigating in vivo cellular targets and optimizing drug delivery strategies for nanomedicines. Key features • This protocol is optimized for investigating nanoparticle uptake in inflamed colonic tissues from DSS-induced colitis models. • This protocol integrates flow cytometry with t-SNE for high-dimensional data analysis, enabling detailed characterization of cellular populations.
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GRP78 Nanobody-Directed Immunotoxin Activates Innate Immunity Through STING Pathway to Synergize Tumor Immunotherapy.
In Adv Sci (Weinh) on 1 May 2025 by Wang, H., Zhou, R., et al.
PubMed
The lack of targetable antigens poses a significant challenge in developing effective cancer-targeted therapies. Cell surface translocation of endoplasmic reticulum (ER) chaperones, such as glucose-regulated protein 78 (GRP78), during malignancy, drug resistance, and ER stress induced by therapies, offers a promising pan-cancer target. To target GRP78, nanobody C5, identified from a phage library and exhibiting high affinity for human and mouse GRP78, is utilized to develop the Pseudomonas exotoxin (PE) immunotoxin C5-PE38. C5-PE38 induced ER stress, apoptosis and immunogenic cell death in targeted cells and showed antitumor efficacy against colorectal cancer and melanoma models without obvious toxicity. Mechanistically, transcriptome profiling showed that C5-PE38 reshaped the tumor immune microenvironment with enhanced innate and adaptive immune response and response to interferon beta. Moreover, C5-PE38-induced cell death could trans-activate STING pathway in dendritic cells and macrophages, promoting CD8+ T cell infiltration. It also sensitizes both primary and metastatic melanomas to anti-PD1 therapy, partly through STING activation. Overall, this study unveils a feasible GRP78 nanobody-directed therapy strategy for single or combinatorial cancer intervention. This work finds that C5-PE38-induced cell death stimulates STING-dependent cytosolic DNA release to promote antitumor immunity, a mechanism not previously reported for PE38, providing valuable insights for its clinical use.
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CDH17-targeting CAR-NK cells synergize with CD47 blockade for potent suppression of gastrointestinal cancers.
In Acta Pharm Sin B on 1 May 2025 by Zheng, L., Ding, Y., et al.
PubMed
Gastrointestinal (GI) cancers are a leading cause of cancer morbidity and mortality worldwide. Despite advances in treatment, cancer relapse remains a significant challenge, necessitating novel therapeutic strategies. In this study, we engineered nanobody-based chimeric antigen receptor (CAR) natural killer (NK) cells targeting cadherin 17 (CDH17) for the treatment of GI tumors. In addition, to enhance the efficacy of CAR-NK cells, we also incorporated CV1, a CD47-SIRPα axis inhibitor, to evaluate the anti-tumor effect of this combination. We found that CDH17-CAR-NK cells effectively eliminated GI cancers cells in a CDH17-dependent manner. CDH17-CAR-NK cells also exhibit potent in vivo anti-tumor effects in cancer cell-derived xenograft and patient-derived xenograft mouse models. Additionally, the anti-tumor activity of CDH17-CAR-NK cells is synergistically enhanced by CD47-signal regulatory protein α (SIRPα) axis inhibitor CV1, likely through augmented macrophages activation and an increase in M1-phenotype macrophages in the tumor microenvironment. Collectively, our findings suggest that CDH17-targeting CAR-NK cells are a promising strategy for GI cancers. The combination of CDH17-CAR-NK cells with CV1 emerges as a potential combinatorial approach to overcome the limitations of CAR-NK therapy. Further investigations are warranted to speed up the clinical translation of these findings.
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Purinergic adipocyte-macrophage crosstalk promotes degeneration of thermogenic brown adipose tissue
In Research Square on 17 April 2025 by Heeren, J., Jaeckstein, M. Y., et al.
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Epidermal Resident Memory T Cell Fitness Requires Antigen Encounter in the Skin
In bioRxiv on 5 April 2025 by Weiss, E. S., Hirai, T., et al.
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Treatment of Acute Liver Injury through Selective Tropism of High Mobility Group Box 1 Gene-Silenced Large Peritoneal Macrophages.
In ACS Nano on 1 April 2025 by Oza, D., Ivich, F., et al.
PubMed
Tissue-resident macrophages (TRMs) are attractive cells to therapeutically deliver oligonucleotide and other gene-expression modifying modalities to treat a wide array of diseases ranging from inflammatory to autoimmune, and even cancer. Here, we focus on TRMs located inside the peritoneal cavity lining the abdomen that selectively express a transcription factor GATA6 called large peritoneal macrophages (GLPMs) and successfully demonstrate functional GLPM-selective delivery of a Cy5-fluorophore-labeled siRNA encapsulated in C12-200 cationic-lipidoid-based nanoparticles (siRNA-Cy5 (C12-200)). Despite being TRMs, GLPMs possess a specific migratory ability to peritoneally located liver tissue upon injury incited by acetaminophen (APAP) overdose in mice. A rapid, liver injury-driven tropism of GLPMs carrying siRNA-Cy5 (C12-200) was seen via systemic circulation, which was elegantly demonstrated by using a noninvasive live-cell tracking technique called diffuse in vivo flow cytometry (DiFC). Finally, RNAi-mediated silencing of a well-known pro-inflammatory damage-associated molecular pattern (DAMP) High Mobility Group Box-1 (HMGB1) gene in GLPMs led to the mitigation of liver injury and inflammation via prevention of GLPM modulation to a pro-inflammatory state, which further translated into significant protection from APAP-driven liver injury and a reduction in liver circulating pro-inflammatory cytokines owing to a muted inflammatory response to acute liver injury. Moreover, silencing HMGB1 by a GalNAc-conjugated hepatocyte-targeting siRNA did not reciprocate the findings, further solidifying our results. Together, our data suggested that GLPMs act as delivery carriers by rapidly bringing lipid nanoparticle-encapsulated RNAi modalities to the injured liver and have emerged as a therapeutically viable strategy to address inflammatory diseases, especially those that are more acute in nature.
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Reprogramming aerobic metabolism mitigates Streptococcus pyogenes tissue damage in a mouse necrotizing skin infection model.
In Nat Commun on 15 March 2025 by Xu, W., Bradstreet, T. R., et al.
PubMed
Disease tolerance is a host response to infection that limits collateral damage to host tissues while having a neutral effect on pathogen fitness. Previously, we found that the pathogenic lactic acid bacterium Streptococcus pyogenes manipulates disease tolerance using its aerobic mixed-acid fermentation pathway via the enzyme pyruvate dehydrogenase, but the microbe-derived molecules that mediate communication with the host's disease tolerance pathways remain elusive. Here we show in a murine model that aerobic mixed-acid fermentation inhibits the accumulation of inflammatory cells including neutrophils and macrophages, reduces the immunosuppressive cytokine interleukin-10, and delays bacterial clearance and wound healing. In infected macrophages, the aerobic mixed-acid fermentation end-products acetate and formate from streptococcal upregulate host acetyl-CoA metabolism and reduce interleukin-10 expression. Inhibiting aerobic mixed-acid fermentation using a bacterial-specific pyruvate dehydrogenase inhibitor reduces tissue damage during murine infection, correlating with increased interleukin-10 expression. Our results thus suggest that reprogramming carbon flow provides a therapeutic strategy to mitigate tissue damage during infection.
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Endogenous antigen processing promotes mRNA vaccine CD4+T cell responses
In bioRxiv on 13 March 2025 by Rood, J. E., Yoon, S. K., et al.
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Lactobacillus rhamnosus GG induces STING-dependent IL-10 in intestinal monocytes and alleviates inflammatory colitis in mice.
In J Clin Invest on 3 February 2025 by Si, W., Zhao, X., et al.
PubMed
Preclinical and clinical observations indicate that the probiotic Lactobacillus rhamnosus GG (LGG) can modulate colonic inflammation. However, the underlying mechanisms have not been explored in depth. Here, we demonstrate that oral administration of live LGG alleviated inflammatory colitis by increasing IL-10 expression in intestinal Ly6C+ monocytes. Mechanistically, LGG induced IL-10 production via the stimulator of IFN genes (STING)/TBK1/NF-κB (RELA) signaling pathway in intestinal Ly6C+ monocytes, enhancing their immune-suppressive function. Elevated IL-10 subsequently activated IL-10 signaling in Ly6C+ monocytes, resulting in an IL-10-based autocrine regulatory loop and inhibition of proinflammatory cytokine production. Furthermore, LGG shifted the gut microbial community and its metabolic functions, leading to intestinal immune responses against colitis. Fecal microbiota transplantation from LGG-colonized mice alleviated immune checkpoint blockade-associated colitis. Our findings highlight the importance of STING signaling in IL-10-dependent antiinflammatory immunity and establish an empirical basis for developing oral administration of live LGG as an efficient and safe therapeutic strategy against inflammatory colitis.
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IL-33-activated ILC2s induce tertiary lymphoid structures in pancreatic cancer.
In Nature on 1 February 2025 by Amisaki, M., Zebboudj, A., et al.
PubMed
Tertiary lymphoid structures (TLSs) are de novo ectopic lymphoid aggregates that regulate immunity in chronically inflamed tissues, including tumours. Although TLSs form due to inflammation-triggered activation of the lymphotoxin (LT)-LTβ receptor (LTβR) pathway1, the inflammatory signals and cells that induce TLSs remain incompletely identified. Here we show that interleukin-33 (IL-33), the alarmin released by inflamed tissues2, induces TLSs. In mice, Il33 deficiency severely attenuates inflammation- and LTβR-activation-induced TLSs in models of colitis and pancreatic ductal adenocarcinoma (PDAC). In PDAC, the alarmin domain of IL-33 activates group 2 innate lymphoid cells (ILC2s) expressing LT that engage putative LTβR+ myeloid organizer cells to initiate tertiary lymphoneogenesis. Notably, lymphoneogenic ILC2s migrate to PDACs from the gut, can be mobilized to PDACs in different tissues and are modulated by gut microbiota. Furthermore, we detect putative lymphoneogenic ILC2s and IL-33-expressing cells within TLSs in human PDAC that correlate with improved prognosis. To harness this lymphoneogenic pathway for immunotherapy, we engineer a recombinant human IL-33 protein that expands intratumoural lymphoneogenic ILC2s and TLSs and demonstrates enhanced anti-tumour activity in PDAC mice. In summary, we identify the molecules and cells of a druggable pathway that induces inflammation-triggered TLSs. More broadly, we reveal a lymphoneogenic function for alarmins and ILC2s.
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The type I IFN-IL-27 axis promotes mRNA vaccine-induced CD8+T cell responses
In bioRxiv on 21 January 2025 by Phan, A. T., Aunins, E., et al.
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Glucocorticoids target postnatal oligodendrocyte precursor cells to modulate adult hippocampal network plasticity and stress-induced behavior
In bioRxiv on 20 December 2024 by Mattioni, L., Poggi, G., et al.
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CD47 predominates over CD24 as a macrophage immune checkpoint in cancer
In bioRxiv on 26 November 2024 by Allen, J., Meglan, A., et al.
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Tumor cell-intrinsic Piezo2 drives radioresistance by impairing CD8+ T cell stemness maintenance.
In J Exp Med on 7 October 2024 by Miao, N., Cao, D., et al.
PubMed
Changes in mechanosensitive ion channels following radiation have seldom been linked to therapeutic sensitivity or specific factors involved in antitumor immunity. Here, in this study, we found that the mechanical force sensor, Piezo2, was significantly upregulated in tumor cells after radiation, and Piezo2 knockout in tumor cells enhanced tumor growth suppression by radiotherapy. Specifically, loss of Piezo2 in tumor cells induced their IL-15 expression via unleashing JAK2/STAT1/IRF-1 axis after radiation. This increase in IL-15 activates IL-15Rα on tumor-infiltrating CD8+ T cells, thereby leading to their augmented effector and stem cell-like properties, along with reduced terminal exhausted feature. Importantly, Piezo2 expression was negatively correlated with CD8 infiltration, as well as with radiosensitivity of patients with rectum adenocarcinoma receiving radiotherapy treatment. Together, our findings reveal that tumor cell-intrinsic Piezo2 induces radioresistance by dampening the IRF-1/IL-15 axis, thus leading to impaired CD8+ T cell-dependent antitumor responses, providing insights into the further development of combination strategies to treat radioresistant cancers.
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Internal regulation between constitutively expressed T cell co-inhibitory receptors BTLA and CD5 and tolerance in recent thymic emigrants.
In Open Biol on 1 October 2024 by Adegoke, A., Thangavelu, G., et al.
PubMed
Immunologic self-tolerance involves signals from co-inhibitory receptors. Several T cell co-inhibitors, including PD-1, are expressed upon activation, whereas CD5 and BTLA are expressed constitutively. The relationship between constitutively expressed co-inhibitors and when they are needed is unknown. Deletion of Btla demonstrated BTLA regulates CD5 expression. Loss of BTLA signals, but not signalling by its ligand, HVEM, leads to increased CD5 expression. Higher CD5 expression set during thymic selection is associated with increased self-recognition, suggesting that BTLA might be needed early to establish self-tolerance. We found that BTLA and PD-1 were needed post-thymic selection in recent thymic emigrants (RTE). RTE lacking BTLA caused a CD4 T cell and MHC class II dependent multi-organ autoimmune disease. Together, our findings identify a negative regulatory pathway between two constitutively expressed co-inhibitors, calibrating their expression. Expression of constitutive and induced co-inhibitory receptors is needed early to establish tolerance in the periphery for RTE.
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YTHDF1 loss in dendritic cells potentiates radiation-induced antitumor immunity via STING-dependent type I IFN production.
In J Clin Invest on 26 September 2024 by Wen, C., Wang, L., et al.
PubMed
The RNA N6-methyladenosine (m6A) reader YTHDF1 is implicated in cancer etiology and progression. We discovered that radiotherapy (RT) increased YTHDF1 expression in dendritic cells (DCs) of PBMCs from patients with cancer, but not in other immune cells tested. Elevated YTHDF1 expression in DCs was associated with poor outcomes for patients receiving RT. We found that loss of Ythdf1 in DCs enhanced the antitumor effects of ionizing radiation (IR) by increasing the cross-priming capacity of DCs across multiple murine cancer models. Mechanistically, IR upregulated YTHDF1 expression in DCs through stimulator of IFN genes/type I IFN (STING/IFN-I) signaling. YTHDF1 in turn triggered STING degradation by increasing lysosomal cathepsins, thereby reducing IFN-I production. We created a YTHDF1 deletion/inhibition prototype DC vaccine that significantly improved the therapeutic effect of RT and radioimmunotherapy in a murine melanoma model. Our findings reveal a layer of regulation between YTHDF1/m6A and STING in response to IR, which opens new paths for the development of YTHDF1-targeting therapies.
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Clustered macrophages cooperate to eliminate tumors via coordinated intrudopodia
In bioRxiv on 20 September 2024 by Dooling, L. J., Anlaş, A. A., et al.
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Protocol for the collection and analysis of the different immune cell subsets in the murine intestinal lamina propria.
In STAR Protoc on 20 September 2024 by Shanmugavadivu, A., Carter, K., et al.
PubMed
The intestinal lamina propria (LP) is a leukocyte-rich cornerstone of the immune system owing to its vital role in immune surveillance and barrier defense against external pathogens. Here, we present a protocol for isolating and analyzing immune cell subsets from the mouse intestinal LP for further downstream applications. Starting from tissue collection and cleaning, epithelium removal, and enzymatic digestion to collection of single cells, we explain each step in detail to maximize the yield of immune cells from the intestinal LP.