InVivoMAb anti-mouse CD16/CD32
Product Details
The 2.4G2 monoclonal antibody reacts specifically with mouse CD16 (FcĪ³RIII) and CD32 (FcĪ³RII). It has also been reported to react non-specifically via its Fc domain to FcĪ³RI. CD16 and CD32 are expressed on B cells, monocytes/macrophages, NK cells, granulocytes, mast cells, and dendritic cells. These receptors bind to the Fc portion of antibody-antigen complexes and play a role in adaptive immune responses. The 2.4G2 antibody is commonly used in flow cytometry and immunofluorescence staining experiments to prevent non-specific binding of the Fc portion of IgG to the FcĪ³III and FcĪ³II, and possibly FcĪ³I, receptors prior to staining with antigen specific primary antibodies. The complete antibody and Fab fragments of the 2.4G2 antibody have also been used to block Fc receptors in vivo. Note that when 2.4G2 is used for Fc blocking in immunoassays and an anti-IgG secondary-step is necessary, the secondary antibody must not be anti-rat IgG2b.Specifications
| Isotype | Rat IgG2b,Ā Īŗ |
|---|---|
| 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 |
<2EU/mg (<0.002EU/Ī¼g) Determined by LAL gel clotting assay |
| Purity |
>95% Determined by SDS-PAGE |
| Sterility | 0.2 Āµm 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 4Ā°C. Do not freeze. |
Additional Formats
Recommended Products
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Recommended Isotype Control(s)
InVivoMAb rat IgG2b isotype control, anti-keyhole limpet hemocyanin
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Recommended Dilution Buffer
InVivoPure pH 8.0 Dilution Buffer
Fc receptor blocking, Flow Cytometry
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 ākiss-and-runā interactions between immune cells āLabelling Immune Partnerships by SorTagging Intercellular Contactsā (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 FcĪ³ receptors (FcĪ³Rs) expressed by host myeloid cells and extend these findings to the human setting. Finally, we demonstrate that in vivo blockade of FcĪ³Rs 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/FcĪ³R interactions that can be modulated to improve checkpoint blockade therapy.
in vivo Fc receptor blocking
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(abā)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(abā)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.
Fc receptor blocking, Flow Cytometry
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.
Fc receptor blocking, Flow Cytometry
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.
Fc receptor blocking, Flow Cytometry
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.
Fc receptor blocking, Flow Cytometry
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.
Fc receptor blocking, Flow Cytometry
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.
Fc receptor blocking, Flow Cytometry
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 receptorās 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, Burkittās 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.
Fc receptor blocking, Flow Cytometry
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.
Fc receptor blocking, Immunofluorescence
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 Ī±4Ī²1 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.
- Mus musculus (House mouse),
- Immunology and Microbiology
Lipid nanoparticle encapsulated large peritoneal macrophages migrate to the lungs via the systemic circulation in a model of clodronate-mediated lung-resident macrophage depletion.
In Theranostics on 22 April 2024 by Oza, D., Ivich, F., et al.
PubMed
Rationale: A mature tissue resident macrophage (TRM) population residing in the peritoneal cavity has been known for its unique ability to migrate to peritoneally located injured tissues and impart wound healing properties. Here, we sought to expand on this unique ability of large peritoneal macrophages (LPMs) by investigating whether these GATA6+ LPMs could also intravasate into systemic circulation and migrate to extra-peritoneally located lungs upon ablating lung-resident alveolar macrophages (AMs) by intranasally administered clodronate liposomes in mice. Methods: C12-200 cationic lipidoid-based nanoparticles were employed to selectively deliver a small interfering RNA (siRNA)-targeting CD-45 labeled with a cyanine 5.5 (Cy5.5) dye to LPMs in vivo via intraperitoneal injection. We utilized a non-invasive optical technique called Diffuse In Vivo Flow Cytometry (DiFC) to then systemically track these LPMs in real time and paired it with more conventional techniques like flow cytometry and immunocytochemistry to initially confirm uptake of C12-200 encapsulated siRNA-Cy5.5 (siRNA-Cy5.5 (C12-200)) into LPMs, and further track them from the peritoneal cavity to the lungs in a mouse model of AM depletion incited by intranasally administered clodronate liposomes. Also, we stained for LPM-specific marker zinc-finger transcription factor GATA6 in harvested cells from biofluids like broncho-alveolar lavage as well as whole blood to probe for Cy5.5-labeled LPMs in the lungs as well as in systemic circulation. Results: siRNA-Cy5.5 (C12-200) was robustly taken up by LPMs. Upon depletion of lung-resident AMs, these siRNA-Cy5.5 (C12-200) labeled LPMs rapidly migrated to the lungs via systemic circulation within 12-24 h. DiFC results showed that these LPMs intravasated from the peritoneal cavity and utilized a systemic route of migration. Moreover, immunocytochemical staining of zinc-finger transcription factor GATA6 further confirmed results from DiFC and flow cytometry, confirming the presence of siRNA-Cy5.5 (C12-200)-labeled LPMs in the peritoneum, whole blood and BALF only upon clodronate-administration. Conclusion: Our results indicate for the very first time that selective tropism, migration, and infiltration of LPMs into extra-peritoneally located lungs was dependent on clodronate-mediated AM depletion. These results further open the possibility of therapeutically utilizing LPMs as delivery vehicles to carry nanoparticle-encapsulated oligonucleotide modalities to potentially address inflammatory diseases, infectious diseases and even cancer. Ā© The author(s).
- In Vitro,
- Mus musculus (House mouse),
- Immunology and Microbiology
Circulating NK cells establish tissue residency upon acute infection of skin and mediate accelerated effector responses to secondary infection.
In Immunity on 9 January 2024 by Torcellan, T., Friedrich, C., et al.
PubMed
Natural killer (NK) cells are present in the circulation and can also be found residing in tissues, and these populations exhibit distinct developmental requirements and are thought to differ in terms of ontogeny. Here, we investigate whether circulating conventional NK (cNK) cells can develop into long-lived tissue-resident NK (trNK) cells following acute infections. We found that viral and bacterial infections of the skin triggered the recruitment of cNK cells and their differentiation into Tcf1hiCD69hi trNK cells that share transcriptional similarity with CD56brightTCF1hi NK cells in human tissues. Skin trNK cells arose from interferon (IFN)-Ī³-producing effector cells and required restricted expression of the transcriptional regulator Blimp1 to optimize Tcf1-dependent trNK cell formation. Upon secondary infection, trNK cells rapidly gained effector function and mediated an accelerated NK cell response. Thus, cNK cells redistribute and permanently position at sites of previous infection via a mechanism promoting tissue residency that is distinct from Hobit-dependent developmental paths of NK cells and ILC1 seeding tissues during ontogeny. Copyright Ā© 2023 The Author(s). Published by Elsevier Inc. All rights reserved.
- Mus musculus (House mouse),
- Biochemistry and Molecular biology,
- Cell Biology,
- Immunology and Microbiology
Reprogramming Short-Chain Fatty Acid Metabolism Mitigates Tissue Damage for Streptococcus pyogenes Necrotizing Skin Infection
Preprint on Research Square on 23 December 2023 by Caparon, M., Xu, W., et al.
PubMed
Disease Tolerance (DT) 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 DT using its aerobic mixed-acid fermentation (ARMAF) pathway via the enzyme pyruvate dehydrogenase (PDH) to alter expression of the immunosuppressive cytokine IL-10. However, the microbe-derived molecules that mediate communication with the hostās DT pathways remain elusive. Here, we show that ARMAF inhibits accumulation of IL-10-producing inflammatory cells including neutrophils and macrophages, leading to delayed bacterial clearance and wound healing. Expression of IL-10 is inhibited through streptococcal production of the short chain fermentation end-products acetate and formate, via manipulation of host acetyl-CoA metabolism, altering non-histone regulatory lysine acetylation. A bacterial-specific PDH inhibitor reduced tissue damage during murine infection, suggesting that reprogramming carbon flow provides a novel therapeutic strategy to mitigate tissue damage during infection.
- Immunology and Microbiology
CTLA-4 antibody-drug conjugate reveals autologous destruction of B-lymphocytes associated with regulatory T cell impairment.
In eLife on 21 December 2023 by Muthana, M. M., Du, X., et al.
PubMed
Germline CTLA-4 deficiency causes severe autoimmune diseases characterized by dysregulation of Foxp3+ Tregs, hyper-activation of effector memory T cells, and variable forms autoimmune cytopenia including gradual loss of B cells. Cancer patients with severe immune-related adverse events (irAE) after receiving anti-CTLA-4/PD-1 combination immunotherapy also have markedly reduced peripheral B cells. The immunological basis for B cell loss remains unexplained. Here, we probe the decline of B cells in human CTLA-4 knock-in mice by using anti-human CTLA-4 antibody Ipilimumab conjugated to a drug payload emtansine (Anti-CTLA-4 ADC). The anti-CTLA-4 ADC-treated mice have T cell hyper-proliferation and their differentiation into effector cells which results in B cell depletion. B cell depletion is mediated by both CD4 and CD8 T cells and at least partially rescued by anti-TNF-alpha antibody. These data revealed an unexpected antagonism between T and B cells and the importance of regulatory T cells in preserving B cells. Ā© 2023, Muthana et al.
- Immunology and Microbiology
IL-23 receptor signaling licenses group 3-like innate lymphoid cells to restrict a live-attenuated oral Chlamydia vaccine in the gut.
In Infection and Immunity on 16 November 2023 by He, Y., Wang, Y., et al.
PubMed
An IFNĪ³-susceptible mutant of Chlamydia muridarum is attenuated in pathogenicity in the genital tract and was recently licensed as an intracellular Oral vaccine vector or intrOv. Oral delivery of intrOv induces transmucosal protection in the genital tract, but intrOv itself is cleared from the gut (without shedding any infectious particles externally) by IFNĪ³ from group 3-like innate lymphoid cells (ILC3s). We further characterized the intrOv interactions with ILC3s in the current study, since the interactions may impact both the safety and efficacy of intrOv as an oral Chlamydia vaccine. Intracolonic inoculation with intrOv induced IFNĪ³ that in return inhibited intrOv. The intrOv-IFNĪ³ interactions were dependent on RORĪ³t, a signature transcriptional factor of ILC3s. Consistently, the transfer of oral intrOv-induced ILC3s from RORĪ³t-GFP reporter mice to IFNĪ³-deficient mice rescued the inhibition of intrOv. Thus, IFNĪ³ produced by intrOv-induced ILC3s is likely responsible for inhibiting intrOv, which is further supported by the observation that oral intrOv did induce significant levels of IFNĪ³-producing LC3s (IFNĪ³+ILC3s). Interestingly, IL-23 receptor knockout (IL-23R-/-) mice no longer inhibited intrOv, which was accompanied by reduced colonic IFNĪ³. Transfer of oral intrOv-induced ILC3s rescued the IL-23R-/- mice to inhibit intrOv, validating the dependence of ILC3s on IL-23R signaling for inhibiting intrOv. Clearly, intrOv induces intestinal IFNĪ³+ILC3s for its own inhibition in the gut, which is facilitated by IL-23R signaling. These findings have provided a mechanism for ensuring the safety of intrOv as an oral Chlamydia vaccine and a platform for investigating how oral intrOv induces transmucosal protection in the genital tract.
- Immunology and Microbiology
Cryptic MHC-E epitope from influenza elicits a potent cytolytic T cell response.
In Nature Immunology on 1 November 2023 by Hogan, M. J., Maheshwari, N., et al.
PubMed
The extent to which unconventional forms of antigen presentation drive T cell immunity is unknown. By convention, CD8 T cells recognize viral peptides, or epitopes, in association with classical major histocompatibility complex (MHC) class I, or MHC-Ia, but immune surveillance can, in some cases, be directed against peptides presented by nonclassical MHC-Ib, in particular the MHC-E proteins (Qa-1 in mice and HLA-E in humans); however, the overall importance of nonclassical responses in antiviral immunity remains unclear. Similarly uncertain is the importance of 'cryptic' viral epitopes, defined as those undetectable by conventional mapping techniques. Here we used an immunopeptidomic approach to search for unconventional epitopes that drive T cell responses in mice infected with influenza virus A/Puerto Rico/8/1934. We identified a nine amino acid epitope, termed M-SL9, that drives a co-immunodominant, cytolytic CD8 T cell response that is unconventional in two major ways: first, it is presented by Qa-1, and second, it has a cryptic origin, mapping to an unannotated alternative reading frame product of the influenza matrix gene segment. Presentation and immunogenicity of M-SL9 are dependent on the second AUG codon of the positive sense matrix RNA segment, suggesting translation initiation by leaky ribosomal scanning. During influenza virus A/Puerto Rico/8/1934 infection, M-SL9-specific T cells exhibit a low level of egress from the lungs and strong differentiation into tissue-resident memory cells. Importantly, we show that M-SL9/Qa-1-specific T cells can be strongly induced by messenger RNA vaccination and that they can mediate antigen-specific cytolysis in vivo. Our results demonstrate that noncanonical translation products can account for an important fraction of the T cell repertoire and add to a growing body of evidence that MHC-E-restricted T cells could have substantial therapeutic value. Ā© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.
- Mus musculus (House mouse),
- Immunology and Microbiology
An essential role for miR-15/16 in Treg suppression and restriction of proliferation.
In Cell Reports on 31 October 2023 by Johansson, K., Gagnon, J. D., et al.
PubMed
The miR-15/16 family targets a large network of genes in TĀ cells to restrict their cell cycle, memory formation, and survival. Upon TĀ cell activation, miR-15/16 are downregulated, allowing rapid expansion of differentiated effector TĀ cells to mediate a sustained response. Here, we used conditional deletion of miR-15/16 in regulatory TĀ cells (Tregs) to identify immune functions of the miR-15/16 family in TĀ cells. miR-15/16 are indispensable to maintain peripheral tolerance by securing efficient suppression by a limited number of Tregs. miR-15/16 deficiency alters expression of critical Treg proteins and results in accumulation of functionally impaired FOXP3loCD25loCD127hi Tregs. Excessive proliferation in the absence of miR-15/16 shifts Treg fate and produces an effector Treg phenotype. These Tregs fail to control immune activation, leading to spontaneous multi-organ inflammation and increased allergic inflammation in a mouse model of asthma. Together, our results demonstrate that miR-15/16 expression in Tregs is essential to maintain immune tolerance. Copyright Ā© 2023 The Author(s). Published by Elsevier Inc. All rights reserved.
- Mus musculus (House mouse)
Commensal bacteria signal through TLR5 and AhR to improve barrier integrity and prevent allergic responses to food.
In Cell Reports on 31 October 2023 by Kemter, A. M., Patry, R. T., et al.
PubMed
The increasing prevalence of food allergies has been linked to reduced commensal microbial diversity. In this article, we describe two features of allergy-protective Clostridia that contribute to their beneficial effects. Some Clostridial taxa bear flagella (a ligand for TLR5) and produce indole (a ligand for the aryl hydrocarbon receptor [AhR]). Lysates and flagella from a Clostridia consortium induced interleukin-22 (IL-22) secretion from ileal explants. IL-22 production is abrogated in explants from mice in which TLR5 or MyD88 signaling is deficient either globally or conditionally in CD11c+ antigen-presenting cells. AhR signaling in RORĪ³t+ cells is necessary for the induction of IL-22. Mice deficient in AhR in RORĪ³t+ cells exhibit increased intestinal permeability and are more susceptible to an anaphylactic response to food. Our findings implicate TLR5 and AhR signaling in a molecular mechanism by which commensal Clostridia protect against allergic responses to food. Copyright Ā© 2023 The Authors. Published by Elsevier Inc. All rights reserved.
- FC/FACS,
- Mus musculus (House mouse),
- Cancer Research,
- Genetics,
- Immunology and Microbiology
Adaptive design of mRNA-loaded extracellular vesicles for targeted immunotherapy of cancer.
In Nature Communications on 19 October 2023 by Dong, S., Liu, X., et al.
PubMed
The recent success of mRNA therapeutics against pathogenic infections has increased interest in their use for other human diseases including cancer. However, the precise delivery of the genetic cargo to cells and tissues of interest remains challenging. Here, we show an adaptive strategy that enables the docking of different targeting ligands onto the surface of mRNA-loaded small extracellular vesicles (sEVs). This is achieved by using a microfluidic electroporation approach in which a combination of nano- and milli-second pulses produces large amounts of IFN-Ī³ mRNA-loaded sEVs with CD64 overexpressed on their surface. The CD64 molecule serves as an adaptor to dock targeting ligands, such as anti-CD71 and anti-programmed cell death-ligand 1 (PD-L1) antibodies. The resulting immunogenic sEVs (imsEV) preferentially target glioblastoma cells and generate potent antitumour activities in vivo, including against tumours intrinsically resistant to immunotherapy. Together, these results provide an adaptive approach to engineering mRNA-loaded sEVs with targeting functionality and pave the way for their adoption in cancer immunotherapy applications. Ā© 2023. Springer Nature Limited.
- Mus musculus (House mouse)
Fibroblast growth factor 18 stimulates the proliferation of hepatic stellate cells, thereby inducing liver fibrosis.
In Nature Communications on 9 October 2023 by Tsuchiya, Y., Seki, T., et al.
PubMed
Liver fibrosis results from chronic liver injury triggered by factors such as viral infection, excess alcohol intake, and lipid accumulation. However, the mechanisms underlying liver fibrosis are not fully understood. Here, we demonstrate that the expression of fibroblast growth factor 18 (Fgf18) is elevated in mouse livers following the induction of chronic liver fibrosis models. Deletion of Fgf18 in hepatocytes attenuates liver fibrosis; conversely, overexpression of Fgf18 promotes liver fibrosis. Single-cell RNA sequencing reveals that overexpression of Fgf18 in hepatocytes results in an increase in the number of Lrat+ hepatic stellate cells (HSCs), thereby inducing fibrosis. Mechanistically, FGF18 stimulates the proliferation of HSCs by inducing the expression of Ccnd1. Moreover, the expression of FGF18 is correlated with the expression of profibrotic genes, such as COL1A1 and ACTA2, in human liver biopsy samples. Thus, FGF18 promotes liver fibrosis and could serve as a therapeutic target to treat liver fibrosis. Ā© 2023. Springer Nature Limited.
- FC/FACS,
- Mus musculus (House mouse),
- Cancer Research,
- Immunology and Microbiology
Activation of melanocortin-1 receptor signaling in melanoma cells impairs T cell infiltration to dampen antitumor immunity.
In Nature Communications on 15 September 2023 by Cui, Y., Miao, Y., et al.
PubMed
Inhibition of T cell infiltration dampens antitumor immunity and causes resistance to immune checkpoint blockade (ICB) therapy. By in vivo CRISPR screening in B16F10 melanoma in female mice, here we report that loss of melanocortin-1 receptor (MC1R) in melanoma cells activates antitumor T cell response and overcomes resistance to ICB. Depletion of MC1R from another melanocytic melanoma model HCmel1274 also enhances ICB efficacy. By activating the GNAS-PKA axis, MC1R inhibits interferon-gamma induced CXCL9/10/11 transcription, thus impairing T cell infiltration into the tumor microenvironment. In human melanomas, high MC1R expression correlates with reduced CXCL9/10/11 expression, impaired T cell infiltration, and poor patient prognosis. Whereas MC1R activation is restricted to melanoma, GNAS activation by hotspot mutations is observed across diverse cancer types and is associated with reduced CXCL9/10/11 expression. Our study implicates MC1R as a melanoma immunotherapy target and suggests GNAS-PKA signaling as a pan-cancer oncogenic pathway inhibiting antitumor T cell response. Ā© 2023. Springer Nature Limited.
Phosphatidylserine clustering by membrane receptors triggers LC3-associated phagocytosis
Preprint on BioRxiv : the Preprint Server for Biology on 7 September 2023 by Boada-Romero, E., Guy, C. S., et al.
PubMed
LC3-associated phagocytosis (LAP) represents a non-canonical function of autophagy proteins in which ATG8 family proteins (LC3 and GABARAP proteins) are lipidated onto single-membrane phagosomes as particles are engulfed by phagocytic cells 1ā4 . LAP plays roles in innate immunity 5 , inflammation and anti-cancer 6 responses and is initiated upon phagocytosis of particles that stimulate Toll-like receptors (TLR), Fc-receptors, and upon engulfment of dying cells 6 . However, how this molecular route is initiated remains elusive. Here we report that receptors that engage LAP enrich phosphatidylserine (PS) in the phagosome membrane via membrane-proximal domains that are necessary and sufficient for LAP to proceed. Subsequently, PS recruits the Rubicon-containing PI3-kinase complex to initiate the enzymatic cascade leading to LAP. Manipulation of plasma membrane PS content, PS-binding by Rubicon, or the PS-clustering domains of receptors prevents LAP and phagosome maturation. We found that pharmacologic inhibition of PS clustering promotes the ability of dendritic cells to induce anti-cancer responses to engulfed tumor cells. Therefore, the initiation of LAP represents a novel mechanism of PS-mediated signal transduction upon ligation of surface receptors.
- Mus musculus (House mouse)
VSIG4 interaction with heparan sulfates inhibits VSIG4-complement binding.
In Glycobiology on 14 August 2023 by Ebstein, S., Rafique, A., et al.
PubMed
V-set and immunoglobulin domain-containing 4 (VSIG4) is a complement receptor of the immunoglobulin superfamily that is specifically expressed on tissue resident macrophages, and its many reported functions and binding partners suggest a complex role in immune function. VSIG4 is reported to have a role in immune surveillance as well as in modulating diverse disease phenotypes such as infections, autoimmune conditions, and cancer. However, the mechanism(s) governing VSIG4's complex, context-dependent role in immune regulation remains elusive. Here, we identify cell surface and soluble glycosaminoglycans, specifically heparan sulfates, as novel binding partners of VSIG4. We demonstrate that genetic deletion of heparan sulfate synthesis enzymes or cleavage of cell-surface heparan sulfates reduced VSIG4 binding to the cell surface. Furthermore, binding studies demonstrate that VSIG4 interacts directly with heparan sulfates, with a preference for highly sulfated moieties and longer glycosaminoglycan chains. To assess the impact on VSIG4 biology, we show that heparan sulfates compete with known VSIG4 binding partners C3b and iC3b. Furthermore, mutagenesis studies indicate that this competition occurs through overlapping binding epitopes for heparan sulfates and complement on VSIG4. Together these data suggest a novel role for heparan sulfates in VSIG4-dependent immune modulation. Ā© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.
- FC/FACS,
- Mus musculus (House mouse),
- Immunology and Microbiology
VĪ³1 and VĪ³4 gamma-delta T cells play opposing roles in the immunopathology of traumatic brain injury in males.
In Nature Communications on 18 July 2023 by Abou-El-Hassan, H., Rezende, R. M., et al.
PubMed
Traumatic brain injury (TBI) is a leading cause of morbidity and mortality. The innate and adaptive immune responses play an important role in the pathogenesis of TBI. Gamma-delta (Ī³Ī“) T cells have been shown to affect brain immunopathology in multiple different conditions, however, their role in acute and chronic TBI is largely unknown. Here, we show that Ī³Ī“ T cells affect the pathophysiology of TBI as early as one day and up to one year following injury in a mouse model. TCRĪ“-/- mice are characterized by reduced inflammation in acute TBI and improved neurocognitive functions in chronic TBI. We find that the VĪ³1 and VĪ³4 Ī³Ī“ T cell subsets play opposing roles in TBI. VĪ³4 Ī³Ī“ T cells infiltrate the brain and secrete IFN-Ī³ and IL-17 that activate microglia and induce neuroinflammation. VĪ³1 Ī³Ī“ T cells, however, secrete TGF-Ī² that maintains microglial homeostasis and dampens TBI upon infiltrating the brain. These findings provide new insights on the role of different Ī³Ī“ T cell subsets after brain injury and lay down the principles for the development of targeted Ī³Ī“ T-cell-based therapy for TBI. Ā© 2023. The Author(s).
- FC/FACS,
- Mus musculus (House mouse),
- Cancer Research
Autologous humanized PDX modeling for immuno-oncology recapitulates features of the human tumor microenvironment.
In Journal for Immunotherapy of Cancer on 1 July 2023 by Chiorazzi, M., Martinek, J., et al.
PubMed
Interactions between immune and tumor cells are critical to determining cancer progression and response. In addition, preclinical prediction of immune-related drug efficacy is limited by interspecies differences between human and mouse, as well as inter-person germline and somatic variation. To address these gaps, we developed an autologous system that models the tumor microenvironment (TME) from individual patients with solid tumors. With patient-derived bone marrow hematopoietic stem and progenitor cells (HSPCs), we engrafted a patient's hematopoietic system in MISTRG6 mice, followed by transfer of patient-derived xenograft (PDX) tissue, providing a fully genetically matched model to recapitulate the individual's TME. We used this system to prospectively study tumor-immune interactions in patients with solid tumor. Autologous PDX mice generated innate and adaptive immune populations; these cells populated the TME; and tumors from autologously engrafted mice grew larger than tumors from non-engrafted littermate controls. Single-cell transcriptomics revealed a prominent vascular endothelial growth factor A (VEGFA) signature in TME myeloid cells, and inhibition of human VEGF-A abrogated enhanced growth. Humanization of the interleukin 6 locus in MISTRG6 mice enhances HSPC engraftment, making it feasible to model tumor-immune interactions in an autologous manner from a bedside bone marrow aspirate. The TME from these autologous tumors display hallmarks of the human TME including innate and adaptive immune activation and provide a platform for preclinical drug testing. Ā© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY. Published by BMJ.
- Mus musculus (House mouse),
- Biochemistry and Molecular biology,
- Cancer Research,
- Cell Biology
Uptake of tumor-derived microparticles induces metabolic reprogramming of macrophages in the early metastatic lung.
In Cell Reports on 27 June 2023 by Kersten, K., You, R., et al.
PubMed
Pre-metastatic niche formation is a critical step during the metastatic spread of cancer. One way by which primary tumors prime host cells at future metastatic sites is through the shedding of tumor-derived microparticles as a consequence of vascular sheer flow. However, it remains unclear how the uptake of such particles by resident immune cells affects their phenotype and function. Here, we show that ingestion of tumor-derived microparticles by macrophages induces a rapid metabolic and phenotypic switch that is characterized by enhanced mitochondrial mass and function, increased oxidative phosphorylation, and upregulation of adhesion molecules, resulting in reduced motility in the early metastatic lung. This reprogramming event is dependent on signaling through the mTORC1, but not the mTORC2, pathway and is induced by uptake of tumor-derived microparticles. Together, these data support a mechanism by which uptake of tumor-derived microparticles induces reprogramming of macrophages to shape their fate and function in the early metastatic lung. Copyright Ā© 2023 The Author(s). Published by Elsevier Inc. All rights reserved.
- Mus musculus (House mouse),
- Immunology and Microbiology
Dynamic chromatin accessibility licenses STAT5- and STAT6-dependent innate-like function of TH9 cells to promote allergic inflammation.
In Nature Immunology on 1 June 2023 by Son, A., Meylan, F., et al.
PubMed
Allergic diseases are a major global health issue. Interleukin (IL)-9-producing helper T (TH9) cells promote allergic inflammation, yet TH9 cell effector functions are incompletely understood because their lineage instability makes them challenging to study. Here we found that resting TH9 cells produced IL-9 independently of T cell receptor (TCR) restimulation, due to STAT5- and STAT6-dependent bystander activation. This mechanism was seen in circulating cells from allergic patients and was restricted to recently activated cells. STAT5-dependent Il9/IL9 regulatory elements underwent remodeling over time, inactivating the locus. A broader 'allergic TH9' transcriptomic and epigenomic program was also unstable. In vivo, TH9 cells induced airway inflammation via TCR-independent, STAT-dependent mechanisms. In allergic patients, TH9 cell expansion was associated with responsiveness to JAK inhibitors. These findings suggest that TH9 cell instability is a negative checkpoint on bystander activation that breaks down in allergy and that JAK inhibitors should be considered for allergic patients with TH9 cell expansion. Ā© 2023. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.
- Mus musculus (House mouse),
- Cancer Research,
- Stem Cells and Developmental Biology
Glioblastoma cell fate is differentially regulated by the microenvironments of the tumor bulk and infiltrative margin.
In Cell Reports on 30 May 2023 by Garcia-Diaz, C., Pƶysti, A., et al.
PubMed
Glioblastoma (GBM) recurrence originates from invasive margin cells that escape surgical debulking, but to what extent these cells resemble their bulk counterparts remains unclear. Here, we generated three immunocompetent somatic GBM mouse models, driven by subtype-associated mutations, to compare matched bulk and margin cells. We find that, regardless of mutations, tumors converge on common sets of neural-like cellular states. However, bulk and margin have distinct biology. Injury-like programs associated with immune infiltration dominate in the bulk, leading to the generation of lowly proliferative injured neural progenitor-like cells (iNPCs). iNPCs account for a significant proportion of dormant GBM cells and are induced by interferon signaling within TĀ cell niches. In contrast, developmental-like trajectories are favored within the immune-cold margin microenvironment resulting in differentiation toward invasive astrocyte-like cells. These findings suggest that the regional tumor microenvironment dominantly controls GBM cell fate and biological vulnerabilities identified in the bulk may not extend to the margin residuum. Copyright Ā© 2023 The Author(s). Published by Elsevier Inc. All rights reserved.
- Mus musculus (House mouse),
- Neuroscience
Sequential Switching Through IgG1 is Redundant for Allergic Reactivity and Memory to Allergens
Preprint on BioRxiv : the Preprint Server for Biology on 28 May 2023 by Koenig, J. F. E., Wade-Vallance, A. K., et al.
PubMed
Allergic reactions to foods are driven by allergen-binding immunoglobulin (Ig)E antibodies. IgE- expressing cells can be generated through a sequential class switching pathway where activated B cells first switch to an intermediary isotype, most frequently IgG1, and then to IgE. It has been proposed that sequential class switch recombination is important in generating high affinity IgE, augmenting anaphylactic reactions, and in holding the memory of IgE responses. Here, we observed surprising redundancy of sequential switching through IgG1 for the functional affinity of the IgE repertoire against multiple food allergens as well as for the ability of IgE to elicit anaphylaxis. We further found that sequential switching via IgG1 was irrelevant for allergic memory. These results indicate that allergen-specific IgG1 B cells are redundant in sensitization, anaphylaxis, and food allergy persistence, thereby implicating other switching pathways as important considerations in the development of therapeutics for allergic diseases.
- FC/FACS,
- Mus musculus (House mouse),
- Cancer Research
STING inhibits the reactivation of dormant metastasis in lung adenocarcinoma.
In Nature on 1 April 2023 by Hu, J., Sanchez-Rivera, F. J., et al.
PubMed
Metastasis frequently develops from disseminated cancer cells that remain dormant after the apparently successful treatment of a primary tumour. These cells fluctuate between an immune-evasive quiescent state and a proliferative state liable to immune-mediated elimination1-6. Little is known about the clearing of reawakened metastatic cells and how this process could be therapeutically activated to eliminate residual disease in patients. Here we use models of indolent lung adenocarcinoma metastasis to identify cancer cell-intrinsic determinants of immune reactivity during exit from dormancy. Genetic screens of tumour-intrinsic immune regulators identified the stimulator of interferon genes (STING) pathway as a suppressor of metastatic outbreak. STING activity increases in metastatic progenitors that re-enter the cell cycle and is dampened by hypermethylation of the STING promoter and enhancer in breakthrough metastases or by chromatin repression in cells re-entering dormancy in response to TGFĪ². STING expression in cancer cells derived from spontaneous metastases suppresses their outgrowth. Systemic treatment of mice with STING agonists eliminates dormant metastasis and prevents spontaneous outbreaks in a T cell- and natural killer cell-dependent manner-these effects require cancer cell STING function. Thus, STING provides a checkpoint against the progression of dormant metastasis and a therapeutically actionable strategy for the prevention of disease relapse. Ā© 2023. The Author(s), under exclusive licence to Springer Nature Limited.
