The2.4G2monoclonal antibody reacts specifically with mouse CD16（FcγRIII）and CD32（FcγRII）。It has also been reported to react non-specifically via its Fc domain to FcγRI.CD16and CD32are 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.The2.4G2antibody is commonly used in flow cytometry and immunofluorescence staining experiments to prevent non-specific binding of the Fc portion of Igto the FG toγIIIIII，I，receptors prior to staining with antigen specific primary antibodies.The complete antibody and Fab fragments of the 2.4 G2 antibody have also been used to block Fc receptorsin vivo.Note that when2.4G2isused for Fc blocking in immunoassays and an anti-IgG secondary-step is necessary，the secondary antibody must not be anti-rat IgG2b。
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 monitoredin vivoby intravital microscopy，this approach does not provide any information on the receptors and ligands involved or enable the isolation of interacting cells fordownstream analysis.Here we describe a complementary approach that uses bacterial sortase A-mediated cell labelling across synapses of immune cells to identify receptor-ligand interangice，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 primingin vivooccur 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 bothin vitroand，andin vivo.Given its flexibility foruse 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。
noclonal antibodies（mAbs）targeting the immune checkpoint anti-programmed cell death protein1（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 mechanism that contribute to their efficicacy or lack thereof.We usedin vivoimaging to uncover the fate and activity of aPD-1mAbs in real time and at subcellular resolution in mice.We show that aPD-1mAbs effectively bind PD-1+tumor-infiltrating CD8+T cells at early time points after administration.However，this engagement is transient，and aPD-1mAbs are captured within minutes from the T cell surface by PD-1-tumor-associated macrophages.We human setting。Finally，we demonstrate thatin vivoblockade of FcγRs before aPD-1mAb administration substantially prolongs aPD-1mAb binding to tumor-infiltrating CD8+T cells and enhances immunotherapy-indced tumor regression in mice.These investigations yield insight into aPD-1target engagementin vivoand identify specific Fc/FcγR interactions that can be modulated to improve checkpoint blockade therapy。
in vivoFCreceptor blocking
Yu，X.，et al.（2015）。“ammonoclonal antibody with anti-D-like activity in murine immune thrombocytopenia requires Fc domain function forimmune thrombocytopenia ameliorative effects”Transfusion55（6Pt2）：1501-1511。PubMed
BACKGROUND:The mechanism of action of anti-Din amelioring 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-Din 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，for，the requirement of Ter119Fc domain in ITP amelioration is investigated using outbred CD-1mice.STUDY DESIGN AND METHODS:Ter119variants，including Ter119F（ab’）2fragments，deglycosylated Ter119，and afucosylated Ter119，were generated to test their effect in amelioration antibody-induced murine ITP。In vivo inhibition of FcgammaRIII and FcgammaRIIB was achieved using the Fab fragment of the FcgammaRIII/FcgammaRIIB-specific MoAb2.4G2.RESULTS:Ter119Fin vitro.Inhibition of FcgammaRIII and FcgammaRIIB，as well as Ter119defucosylation，do not affect Ter119-mediated ITP amelioration.CONCLUSION:The Fc domain of Ter119，as well asits Fc glycosylation，is required for Ter119-mediated ITP amelioration.Moreover，both Fc and Fc glycosylation are required for Ter119-mediated phagytosisin 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）。“CD47blockade triggers T cell-mediated destruction of immunogenic tumors”Nat Med21（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 CD47blockade depend on dendritic cell but not macrophage cross-priming of T cell responses.The therapeutic effects of anti-CD47antibody therapy were abrogated in T cell-deficient mice.In addition，the antitumor effects of CD47blockaderequired expression of DNA，but neither MyD88nor 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 timining of administration of standard chemotherapy markedly impacted the induction of antitumor T cell responses by CD47blockade.Together，our findings indicate that CD47blockade 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 Commun6:7114。PubMed
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，mechanism 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 signtifitureture mothy intratumoral activation.Development of this vasculature is controlled by a mechanism involving effector CD8T cells and NK cells that secrete LTalpha3and IFNgamma.LN-like vasculature is also associated with organized aggregates of blymphocytes 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。
IL-17-producing CD4（+）T（Th17）cells，along with IFN-gamma-expressing Th1cells，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 Th1and Th17cells have been largely characterized.Among them，IL-23 is essential for the generation of stable and encephalitogenic Th17cells and for the development of EAE.The IL-7/IL-7R signaling axis parcicells，in，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 Th17cells from mice with EAE express high levels of IL-7 Ralpha compared with Th1cells.Using mice that constitutively express IL-7 Ralpha on T cells，we determined that sustained IL-7 rexpression in IL-23R-deficient mice could not drive pathenic T cells and EAp7 hibited the differentation of Th17cells，but promoted IFN-gamma and GM-CSF secretionin vitro.In vivo IL-7/anti-IL-7mAb complexes selectively expanded and enhanced the proliferation of CXCR3-expressing Th1 cells，but did not impact Th17cells 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 Th17cells to IFN-gamma-producing T cells.Together，these data address the contribution of IL-23/IL-23R and IL-7/IL-7R signaling in Th17and Th1cell 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-2availability”Nat Commun5:3495。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 aprecursor-progeny relationship between Tregs and Tfh cells，but are instead mediated by increased availability of IL-2，wch hippressses the differeation and centiation of，compromises the GC B response.Thus，Tregs promote influenza-specific GC responses by preventing excessive IL-2signalling，which suppresses Tfh cell differentiation。
Fc receptor blocking，Flow Cytometry
Deng，L.，et al.（2014）。“Irradiation and anti-PD-L1treatment synergistically promote antitumor immunity in mice”J Clin Invest124（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-ligand1（PD-L1，also called B7-H1）can enhance T cell effector function when PD-L1is expressed in chronically inflamed tissues and tumors.Here，we demonstrate that PD-L1was upregulated in the tumor microenvironment after IR.Administration of anti-PD-L1enhanced the efficacy of IR through acytotoxic 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 action between IR，T cells，and the PD-L1/PD-1axis and establish a basis for the rational design of combination therapy with immune modulators and radiotherapy。
Fc receptor blocking，Flow Cytometry
uppidi，J.R.，et al.（2014）。“Loss of signalling via Galpha13in germinal centre B-cell-derived lymphoma”Nature516（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），a Galpha12and Galpha13coupled 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）andS1PR2（refs5,6,7）。炉膛，炉膛in vitroand，andin vivoassays，that GCB-DLBCL-associated mutations occurring in S1PR2frequently 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 S1，P，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 Galpha13effector ARHGEF1，and Arhgef1 deficiency also led to germinal centre B-cell dissemination.The incomplete phenocopy of Galpha13-and S1PR2deficiency单击功能区上，anorphan receptor that is mutated in GCB-DLBCL and another germinal centre B-cell-derived malignancy，Burkitt’slymphoma，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 lma。
Fc receptor blocking，Flow Cytometry
Heesch，K.，et al.（2014）。“The function of the chemokine receptor CXCR6in the T cell response of mice against Listeria monocytogenes”PLoS One9（5）：e97701。PubMed
The chemokine receptor CXCR6is expressed on different T cell subsets and up-regulated following T cell activation.CXCR6has been implicated in the localization of cells to the liver due to the constitutive expression of its ligand CXCL16on liver sinusoidal endothelial cells.Here，we analyzed the role of CXCR6in 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 CXCR6did 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，CXCR6was dispensable at later time points of the CD8+T cell response.When transferred CD8+T cells were folllowed 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 CXCR6is not required for the formation of atcell response to L.monocytogenes and for the accumulation of T cells in the infected liver but CXCR6appears 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 Immunol191（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 andα4β1integrin，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.CD8memory T cells in LN that express these moleculs 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 scrientscectry of chanisms。
