Paired Ig-like Receptor Research Articles

Alloantigen Infusion Activates the Transcriptome of Type 2 Conventional Dendritic Cells.

Recent studies have revealed novel molecular mechanisms by which innate monocytic cells acutely recognize and respond to alloantigen with significance to allograft rejection and tolerance. What remains unclear is the single-cell heterogeneity of the innate alloresponse, particularly the contribution of dendritic cell (DC) subsets. To investigate the response of these cells to exposure of alloantigen, C57BL/6J mice were administered live allogenic BALB/cJ splenic murine cells versus isogenic cells. In parallel, we infused apoptotic allogenic and isogenic cells, which have been reported to modulate immunity. Forty-eight hours after injection, recipient spleens were harvested, enriched for DCs, and subjected to single-cell mRNA sequencing. Injection of live cells induced a greater transcriptional change across DC subsets compared with apoptotic cells. In the setting of live cell infusion, type 2 conventional DCs (cDC2s) were most transcriptionally responsive with a Ccr2+ cDC2 subcluster uniquely responding to the presence of alloantigen compared with the isogenic control. In vitro experimentation confirmed unique activation of CCR2+ cDC2s following alloantigen exposure. Candidate receptors of allorecognition in other innate populations were interrogated and A type paired Ig-like receptors were found to be increased in the cDC2 population following alloexposure. These results illuminate previously unclear distinctions between therapeutic infusions of live versus apoptotic allogenic cells and suggest a role for cDC2s in innate allorecognition. More critically, these studies allow for future interrogation of the transcriptional response of immune cells in the setting of alloantigen exposure invivo, encouraging assessment of novel pathways and previously unexamined receptors in this setting.

ANGPTL8 is a negative regulator in pathological cardiac hypertrophy

Pathological cardiac hypertrophy is an independent risk factor for heart failure and is considered a target for the treatment of heart failure. However, the mechanisms underlying pathological cardiac hypertrophy remain largely unknown. We aimed to investigate the role of angiopoietin-like protein 8 (ANGPTL8) in pathological cardiac hypertrophy. We found that serum ANGPTL8 levels were significantly increased in hypertensive patients with cardiac hypertrophy and in mice with cardiac hypertrophy induced by Ang II or TAC. Furthermore, the secretion of ANGPTL8 from the liver was increased during hypertrophic processes, which were triggered by Ang II. In the Ang II- and transverse aortic constriction (TAC)-induced mouse cardiac hypertrophy model, ANGPTL8 deficiency remarkably accelerated cardiac hypertrophy and fibrosis with deteriorating cardiac dysfunction. Accordingly, both recombinant human full-length ANGPTL8 (rANGPTL8) protein and ANGPTL8 overexpression significantly mitigated Ang II-induced cell enlargement in primary neonatal rat cardiomyocytes (NRCMs) and H9c2 cells. Mechanistically, the antihypertrophic effects of ANGPTL8 depended on inhibiting Akt and GSK-3β activation, and the Akt activator SC-79 abolished the antihypertrophic effects of rANGPTL8 in vitro. Moreover, we demonstrated that ANGPTL8 directly bound to the paired Ig-like receptor PIRB (LILRB3) by RNA-seq and immunoprecipitation-mass screening. Remarkably, the antihypertrophic effects of ANGPTL8 were largely blocked by anti-LILRB3 and siRNA-LILRB3. Our study indicated that ANGPTL8 served as a novel negative regulator of pathological cardiac hypertrophy by binding to LILRB3 (PIRB) and inhibiting Akt/GSK3β activation, suggesting that ANGPTL8 may provide synergistic effects in combination with AT1 blockers and become a therapeutic target for cardiac hypertrophy and heart failure.

ILT4 in Colorectal Cancer Cells Induces Suppressive T Cell Contexture and Disease Progression.

PurposeImmune checkpoint blockade (ICB) therapy shows little or no clinical benefit in most colorectal cancer (CRC) patients, due to the immunosuppressive T cell contexture in the tumor microenvironment (TME). Immunoglobulin-like transcript (ILT) 4 is an immunosuppressive molecule in myeloid cells. ILT4 is enriched in solid tumor cells, facilitating their proliferation, invasion, and metastasis. However, the regulatory role of ILT4 in T cell immunity of CRC is still undetermined. Here, we aimed to explore how tumor cell-derived ILT4 orchestrates T cell infiltration, subset distribution, and function in CRC.MethodsA total of 145 paraffin-embedded cancer tissues and the corresponding clinicopathological information were collected from CRC patients. Immunohistochemical (IHC) staining and public database analyses determined the correlation of ILT4 expression with different T cell subset densities, IFN-γ levels, and patient outcomes. Paired Ig-like receptor B (PIR-B, ILT4 mouse ortholog)-overexpressing/-downregulated MC38 cells were subcutaneously injected into C57BL/6 mice as a CRC transplantation model. The frequencies, subsets, and IFN-γ levels of T cells in mouse blood and spleens were determined using flow cytometry and immunohistochemistry, respectively.ResultsHigh ILT4 expression in CRC cells was associated with decreased T cell infiltration, disease progression, and poor patient survival. T cell subset analyses indicated that ILT4-high patients showed reduced CD8+ T cell but elevated FOXP3+ regulatory T (Treg) cell frequencies in the TME. High ILT4 levels predicted lower IFN-γ production by tumor-infiltrating lymphocytes (TILs), especially by CD8+T cells in human CRC tissues. Moreover, PIR-B overexpression accelerated MC38 growth in mice, decreased CD3+/CD8+/IFN-γ+ T cell densities, and elevated Treg infiltration in the TME, blood, and spleens. PIR-B knockdown had the opposite effects.ConclusionILT4 in CRC cells induced immunosuppressive T cell subset infiltration and impaired IFN-γ production in TILs, suggesting that ILT4 might be a potential immunotherapeutic target and prognostic biomarker.

ILT4 inhibition prevents TAM- and dysfunctional T cell-mediated immunosuppression and enhances the efficacy of anti-PD-L1 therapy in NSCLC with EGFR activation.

Rationale: Immune checkpoint inhibitors (ICIs) against the PD-1/PD-L1 pathway showed limited success in non-small cell lung cancer (NSCLC) patients, especially in those with activating epidermal growth factor receptor (EGFR) mutations. Elucidation of the mechanisms underlying EGFR-mediated tumor immune escape and the development of effective immune therapeutics are urgently needed. Immunoglobulin-like transcript (ILT) 4, a crucial immunosuppressive molecule initially identified in myeloid cells, is enriched in solid tumor cells and promotes the malignant behavior of NSCLC. However, the upstream regulation of ILT4 overexpression and its function in tumor immunity of NSCLC with EGFR activation remains unclear.Methods: ILT4 expression and EGFR phosphorylation in human NSCLC tissues and cell lines were analyzed using immunohistochemistry (IHC), real-time PCR, Western blotting, immunofluorescence, and flow cytometry. The molecular signaling for EGFR-regulated ILT4 expression was investigated using mRNA microarray and The Cancer Genome Atlas (TCGA) database analyses and then confirmed by Western blotting. The regulation of tumor cell proliferation and apoptosis by ILT4 was examined by CCK8 proliferation and apoptosis assays. The impact of ILT4 and PD-L1 on tumor-associated macrophage (TAM) recruitment and polarization was evaluated using Transwell migration assay, flow cytometry, enzyme linked immunosorbent assay (ELISA) and real-time PCR, while their impact on T cell survival and cytotoxicity was analyzed by CFSE proliferation assay, apoptotic assay, flow cytometry, ELISA and cytolytic assay. Tumor immunotherapy models targeting at paired Ig-like receptor B (PIR-B, an ortholog of ILT4 in mouse)/ILT4 and/or PD-L1 were established in C57BL/6 mice inoculated with stable EGFR- overexpressing Lewis lung carcinoma (LLC) cells and in humanized NSG mice inoculated with EGFR mutant, gefitinib-resistant PC9 (PC9-GR) or EGFR-overexpressing wild type H1299 cells. PIR-B and ILT4 inhibition was implemented by infection of specific knockdown lentivirus and PD-L1 was blocked using human/mouse neutralizing antibodies. The tumor growth model was established in NSG mice injected with PIR-B-downregulated LLC cells to evaluate the effect of PIR-B on tumor proliferation. The frequencies and phenotypes of macrophages and T cells in mouse spleens and blood were detected by flow cytometry while those in tumor tissues were determined by IHC and immunofluorescence.Results: We found that ILT4 expression in tumor cells was positively correlated with EGFR phosphorylation in human NSCLC tissues. Using NSCLC cell lines, we demonstrated that ILT4 was upregulated by both tyrosine kinase mutation-induced and epidermal growth factor (EGF)-dependent EGFR activation and subsequent AKT/ERK1/2 phosphorylation. Overexpressed ILT4 in EGFR-activated tumor cells induced TAM recruitment and M2-like polarization, which impaired T cell function. ILT4 also directly inhibited T cell proliferation, cytotoxicity, and IFN-γ expression and secretion. In EGFR-activated cell lines in vitro and in wild-type EGFR-activated C57BL/6 and humanized NSG immunotherapy models in vivo, either ILT4 (PIR-B) or PD-L1 inhibition enhanced anti-tumor immunity and suppressed tumor progression by counteracting TAM- and dysfunctional T cell- induced immuno-suppressive TME; the combined inhibition of both molecules showed the most dramatic tumor retraction. Surprisingly, in EGFR mutant, TKI resistant humanized NSG immunotherapy model, ILT4 inhibition alone rather than in combination with a PD-L1 inhibitor suppressed tumor growth and immune evasion.Conclusions: ILT4 was induced by activation of EGFR-AKT and ERK1/2 signaling in NSCLC cells. Overexpressed ILT4 suppressed tumor immunity by recruiting M2-like TAMs and impairing T cell response, while ILT4 inhibition prevented immunosuppression and tumor promotion. Furthermore, ILT4 inhibition enhanced the efficacy of PD-L1 inhibitor in EGFR wild-type but not in EGFR mutant NSCLC. Our study identified novel mechanisms for EGFR-mediated tumor immune escape, and provided promising immunotherapeutic strategies for patients with EGFR-activated NSCLC.

Therapeutic effects of soluble human leukocyte antigen G2 isoform in lupus-prone MRL/lpr mice

Human leukocyte antigen (HLA)-G, a non-classical HLA class I molecule, has one of the splicing isoforms, HLA-G2, which lacks one domain (α2) and forms a non-covalent homodimer. HLA-G2 is expressed on placental cells, regulatory T cells, tumor cells, and virus-infected cells, and is involved in immunosuppression. The major isoform of HLA-G, HLA-G1, binds to leukocyte immunoglobulin (Ig)-like receptor (LILR) B1 and LILRB2, on the contrary, HLA-G2 binds to only LILRB2. We previously reported that HLA-G2 bound LILRB2 more strongly than HLA-G1 and also to paired Ig-like receptor (PIR)-B, a mouse homolog of LILRBs. Furthermore, HLA-G2 showed immunosuppressive effects in both collagen-induced arthritis (CIA) and atopic dermatitis-like model mice. In this study, we examine in vivo effects of HLA-G2 in systemic lupus erythematosus (SLE) model mice. HLA-G2 showed the suppression of the typical SLE symptoms such as serum anti-dsDNA antibody level and urinary albumin index. Furthermore, HLA-G2 tended to downregulate B-lymphocyte stimulator (BLyS) production. This is the first observation of the immunosuppressive effects of HLA-G2 isoform in SLE model mice, suggesting that HLA-G2 could be a useful therapeutic agent for SLE.

Glatiramer Acetate Enhances Myeloid-Derived Suppressor Cell Function via Recognition of Paired Ig-like Receptor B.

Glatiramer acetate (GA; Copaxone) is a copolymer therapeutic that is approved by the Food and Drug Administration for the relapsing-remitting form of multiple sclerosis. Despite an unclear mechanism of action, studies have shown that GA promotes protective Th2 immunity and stimulates release of cytokines that suppress autoimmunity. In this study, we demonstrate that GA interacts with murine paired Ig-like receptor B (PIR-B) on myeloid-derived suppressor cells and suppresses the STAT1/NF-κB pathways while promoting IL-10/TGF-β cytokine release. In inflammatory bowel disease models, GA enhanced myeloid-derived suppressor cell-dependent CD4+ regulatory T cell generation while reducing proinflammatory cytokine secretion. Human monocyte-derived macrophages responded to GA by reducing TNF-α production and promoting CD163 expression typical of alternative maturation despite the presence of GM-CSF. Furthermore, GA competitively interacts with leukocyte Ig-like receptors B (LILRBs), the human orthologs of PIR-B. Because GA limited proinflammatory activation of myeloid cells, therapeutics that target LILRBs represent novel treatment modalities for autoimmune indications.

Regulation of immune and neural function via leukocyte Ig-like receptors.

Leukocyte Ig-like receptors (LILRs)/Ig-like transcripts (ILTs) are expressed on innate and adaptive immune cells and maintain immune homeostasis. LILRs consist of activating and inhibitory-type receptors that regulate adequate cellular functions. LILRs were firstly identified as MHC class I receptors, therefore expression and/or polymorphisms of LILRs are reported to associate with autoimmune disorders and transplant rejection; however, recent accumulating evidences have revealed that LILRs recognize with diverse ligands including bacteria and virus. In addition, inhibitory LILRB2 (ILT4) and murine relative paired Ig-like receptor (PIR)-B are expressed on neuron and is involved in the dysregulation of central nervous system via interaction with neuronal ligands including amyloid β-protein. In this review, we summarize recent discoveries on the functions of inhibitory MHC class I receptors, and discuss their regulatory roles in immune responses and neural functions.

Cell-Autonomous Regulation of Dendritic Spine Density by PirB.

Synapse density on cortical pyramidal neurons is modulated by experience. This process is highest during developmental critical periods, when mechanisms of synaptic plasticity are fully engaged. In mouse visual cortex, the critical period for ocular dominance (OD) plasticity coincides with the developmental pruning of synapses. At this time, mice lacking paired Ig-like receptor B (PirB) have excess numbers of dendritic spines on L5 neurons; these spines persist and are thought to underlie the juvenile-like OD plasticity observed in adulthood. Here we examine whether PirB is required specifically in excitatory neurons to exert its effect on dendritic spine and synapse density during the critical period. In mice with a conditional allele of PirB (PirBfl/fl), PirB was deleted only from L2/3 cortical pyramidal neurons in vivo by timed in utero electroporation of Cre recombinase. Sparse mosaic expression of Cre produced neurons lacking PirB in a sea of wild-type neurons and glia. These neurons had significantly elevated dendritic spine density, as well as increased frequency of miniature EPSCs, suggesting that they receive a greater number of synaptic inputs relative to Cre- neighbors. The effect of cell-specific PirB deletion on dendritic spine density was not accompanied by changes in dendritic branching complexity or axonal bouton density. Together, results imply a neuron-specific, cell-autonomous action of PirB on synaptic density in L2/3 pyramidal cells of visual cortex. Moreover, they are consistent with the idea that PirB functions normally to corepress spine density and synaptic plasticity, thereby maintaining headroom for cells to encode ongoing experience-dependent structural change throughout life.

Paired Ig-like Receptor B Inhibits IL-13-Driven Eosinophil Accumulation and Activation in the Esophagus.

Eosinophilic esophagitis (EoE) is a Th2 cytokine-associated disease characterized by eosinophil infiltration, epithelial cell hyperplasia, and tissue remodeling. Recent studies highlighted a major contribution for IL-13 in EoE pathogenesis. Paired Ig-like receptor B is a cell surface immune-inhibitory receptor that is expressed by eosinophils and postulated to regulate eosinophil development and migration. We report that Pirb is upregulated in the esophagus after inducible overexpression of IL-13 (CC10-Il13(Tg) mice) and is overexpressed by esophageal eosinophils. CC10-Il13(Tg)/Pirb(-/-) mice displayed increased esophageal eosinophilia and EoE pathology, including epithelial cell thickening, fibrosis, and angiogenesis, compared with CC10-Il13(Tg)/Pirb(+/+) mice. Transcriptome analysis of primary Pirb(+/+) and Pirb(-/-) esophageal eosinophils revealed increased expression of transcripts associated with promoting tissue remodeling in Pirb(-/-) eosinophils, including profibrotic genes, genes promoting epithelial-to-mesenchymal transition, and genes associated with epithelial growth. These data identify paired Ig-like receptor B as a molecular checkpoint in IL-13-induced eosinophil accumulation and activation, which may serve as a novel target for future therapy in EoE.

The immunosuppressive effect of domain-deleted dimer of HLA-G2 isoform in collagen-induced arthritis mice

HLA-G is involved in maternal-fetal immune tolerance and is reported to be a natural tolerogenic molecule. Seven-spliced isoforms including dimeric and β2m-free forms have been identified. The major isoform, HLA-G1 (and its soluble type HLA-G5), binds to the inhibitory immune receptors, leukocyte immunoglobulin (Ig)-like receptor (LILR) B1 and LILRB2. We previously reported that HLA-G1 also binds to paired Ig-like receptor (PIR)-B, a mouse homolog of LILRBs, and had a significant immunosuppressive effect in collagen-induced arthritis (CIA) mice. Although HLA-G2 and its soluble form HLA-G6 bind specifically to LILRB2, its functional characteristics are largely unknown. In this study, we report the significant immunosuppressive effect of HLA-G2 dimer in CIA mice. Surface plasmon resonance analysis revealed a specific interaction of HLA-G2 with PIR-B. CIA mice were administered HLA-G2 protein subcutaneously once in the left footpad and clinical severity was evaluated in a double-blind study. A single administration of HLA-G2 maintained a suppressive effect for over 1month. These results suggested that the HLA-G2 protein might be a useful biopharmaceutical for the treatment of rheumatoid arthritis by binding to inhibitory PIR-B.

A bone to pick with Fc gamma receptors.

Bone formation is a dynamic process, in which the bone structure is constantly remodeled. Osteoclasts and osteoblasts play critical but opposing roles in bone formation and resorption. While osteoclasts promote bone resorption, osteoblasts drive bone formation. Both processes are intertwined and tightly regulated to ensure the integrity of the bony skeleton. In particular, bone mass, strength and mineral homeostasis depend on balanced osteoclast and osteoblast function. Enhanced osteoclast activity leads to massive bone loss as exemplified in autoimmune diseases like rheumatoid arthritis (RA) or systemic lupus erythematosus (SLE). Osteoclasts and osteoblasts originate from different precursors. Whereas osteoblasts derive from mesenchymal stem cells, osteoclasts originate from multinucleated progenitors of the monocyte/ macrophage family. Two critical factors that regulate osteoclastogenesis are macrophage colony-stimulating factor (M-CSF) and receptor for activation of nuclear factor kappa B (NF-κB) (RANK) ligand (RANKL). RANKL is expressed by T cells, endothelial cells and osteoblasts. Although activation of the RANKL pathway is essential to initiate osteoclastogenesis, an immunoreceptor tyrosin based activation motif (ITAM) co-stimulatory pathway is required for calcium-mediated activation of nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), which serves as an important factor in osteoclast differentiation. The ITAM co-stimulatory pathway is activated in response to ligation of immunoglobulin-like receptors such as osteoclast-associated receptor (OSCAR), triggering receptor expressed on myeloid cells (TREM-2) or paired Ig-like receptor A (PIR-A), and the phosphorylation of the adaptor molecules containing ITAM motifs. Such ITAM motif-containing proteins are DNAX activation protein of 12 kDa (DAP12) and the Fc-receptor γ subunit (FcRγ) Importantly, the γ-chain not only facilitates FcγR signaling but is also required for the transport of IgG Fc receptors to the cell surface. The critical role of FcRγ and DAP12 for osteoclast activation was first demonstrated in mice suffering from severe osteopetrosis when both factors were lacking. Importantly, the phenotype was less pronounced in mice lacking only DAP12, whereas FcRγ-deficient mice showed no disease phenotype. These data suggest that FcRγ plays an important role in osteoclastogenesis in concert with DAP12.

Dual Effects of IL-1 Overactivity on the Immune System in a Mouse Model of Arthritis due to Deficiency of IL-1 Receptor Antagonist

Previous studies have revealed the significance of cytokine interleukin 1 (IL-1) in the onset and progression of rheumatoid arthritis (RA). The precise molecular mechanisms related to IL-1 underlying RA is still elusive. We conducted a whole genome-wide transcriptomal comparison of wild-type (WT) and arthritis-prone IL-1 receptor antagonist (IL-1rn) deficient BALB/c mice to address this issue. To refine our search efforts, gene expression profiling was also performed on paired wild-type and arthritis-resistant IL-1rn deficient DBA/1 mice as internal controls when identifying causative arthritis candidate genes. Two hundred and fifteen transcripts were found to be dysregulated greater than or equal to 2-fold in the diseased mice. The altered transcriptome in BALB/c mice revealed increased myeloid cell activities and impaired lymphocyte functionality, suggesting dual regulatory effects of IL-1 hyperactivity on immunological changes associated with arthritis development. Phase-specific gene expression changes were identified, such as early increase and late decrease of heat shock protein coding genes. Moreover, common gene expression changes were also observed, especially the upregulation of paired Ig-like receptor A (Pira) in both early and late phases of arthritis. Real-time PCR was performed to validate the expression of Pira and an intervention experiment with a major histocompatibility complex (MHC) class I inhibitor (brefeldin A) was carried out to investigate the role of suppressing Pira activity. We conclude that global pattern changes of common and distinct gene expressions may represent novel opportunities for better control of RA through early diagnosis and development of alternative therapeutic strategies.

Inhibitory Receptor Paired Ig-like Receptor B Is Exploited by Staphylococcus aureus for Virulence

The innate immune system has developed to acquire a wide variety of pattern-recognition receptors (PRRs) to identify potential pathogens, whereas pathogens have also developed to escape host innate immune responses. ITIM-bearing receptors are attractive targets for pathogens to attenuate immune responses against them; however, the in vivo role of the inhibitory PRRs in host-bacteria interactions remains unknown. We demonstrate in this article that Staphylococcus aureus, a major Gram-positive bacteria, exploits inhibitory PRR paired Ig-like receptor (PIR)-B on macrophages to suppress ERK1/2 and inflammasome activation, and subsequent IL-6 and IL-1β secretion. Consequently, Pirb(-/-) mice infected with S. aureus showed enhanced inflammation and more effective bacterial clearance, resulting in resistance to the sepsis. Screening of S. aureus mutants identified lipoteichoic acid (LTA) as an essential bacterial cell wall component required for binding to PIR-B and modulating inflammatory responses. In vivo, however, an LTA-deficient S. aureus mutant was highly virulent and poorly recognized by macrophages in both wild-type and Pirb(-/-) mice, demonstrating that LTA recognition by PRRs other than PIR-B mediates effective bacterial elimination. These results provide direct evidence that bacteria exploit the inhibitory receptor for virulence, and host immune system counterbalances the infection.

Characterisation of Bovine Leukocyte Ig-like Receptors

Leukocyte Immunoglobulin-like receptors (LILR) are innate immune receptors involved in regulating both innate and adaptive immune functions. LILR show more interspecies conservation than the closely related Killer Ig-like receptors, and homologues have been identified in rodents, primates, seals and chickens. The murine equivalents, paired Ig-like receptors (PIR), contain two additional immunoglobulin domains, but show strong sequence and functional similarities to human LILR. The bovine genome was recently sequenced, with preliminary annotations indicating that LILR were present in this species. We therefore sought to identify and characterize novel LILR within the Bos taurus genome, compare these phylogenetically with LILR from other species and determine whether they were expressed in vivo. Twenty six potential bovine LILR were initially identified using BLAST and BLAT software. Phylogenetic analysis constructed using the neighbour-joining method, incorporating pairwise deletion and confidence limits estimated from 1000 replicates using bootstrapping, indicated that 16 of these represent novel bovine LILR. Protein structures defined using protein BLAST predict that the bovine LILR family comprises seven putative inhibitory, four activating and five soluble receptors. Preliminary expression analysis was performed by mapping the predicted sequences with raw data from total transcript sequence generated using Genome Analyzer IIx (Illumina) to provide evidence that all 16 of these receptors are expressed in vivo. The bovine receptor family appears to contain receptors which resemble the six domain rodent PIR as well as the four domain LILR found in other species.

Transcriptional Activation of the Pirb Gene in B Cells by PU.1 and Runx3

Cells in the immune system are regulated positively or negatively by sets of receptor pairs that conduct balanced, activating, or inhibitory intracellular signaling. One such receptor pair termed paired Ig-like receptor (PIR) is composed of the inhibitory PIR-B and its activating isoform, PIR-A. Upon binding to their shared ligand, MHC class I molecules, these receptors control the threshold for immune cell activation. Gene-targeting studies on PIR-B in mice revealed the importance of the inhibition mediated by the PIR-B-MHC interaction in the immune system. Recent studies also revealed the significance of the interaction of PIR-B with neurite outgrowth inhibitors, including Nogo in the CNS. The coordinated regulation by PIR-B and PIR-A is considered to be primarily dependent on their expression balance in cells. However, the mechanism underlying transcriptional control of the genes for PIR-B and PIR-A (Pirb and Pira, respectively) remains to be clarified. In this study, we identified the major cis-acting promoter segment for Pirb and Pira in B cells as the -212 to -117 region upstream from the translation initiation codon. PU.1 and Runx3 were found to bind to this Pirb promoter. Truncation of the PU.1-binding motif significantly reduced the promoter activity, whereas the influence of elimination of the Runx3 site was marginal in B lymphoma BCL1-B20 cells. Unexpectedly, PU.1, but not Runx3, knockdown reduced the levels of both the Pirb and Pira transcripts. We conclude that the major promoter of Pirb, and probably Pira as well, is activated dominantly by PU.1 and marginally by Runx3 in B cells.

Immunoinhibitory Adapter Protein Src Homology Domain 3 Lymphocyte Protein 2 (SLy2) Regulates Actin Dynamics and B Cell Spreading

Appropriate B cell activation is essential for adaptive immunity. In contrast to the molecular mechanisms that regulate positive signaling in immune responses, the counterbalancing negative regulatory pathways remain insufficiently understood. The Src homology domain 3 (SH3)-containing adapter protein SH3 lymphocyte protein 2 (SLy2, also known as hematopoietic adapter-containing SH3 and sterile α-motif (SAM) domains 1; HACS1) is strongly up-regulated upon B cell activation and functions as an endogenous immunoinhibitor in vivo, but the underlying molecular mechanisms of SLy2 function have been elusive. We have generated transgenic mice overexpressing SLy2 in B and T cells and have studied the biological effects of elevated SLy2 levels in Jurkat and HeLa cells. Our results demonstrate that SLy2 induces Rac1-dependent membrane ruffle formation and regulates cell spreading and polarization and that the SLy2 SH3 domain is essential for these effects. Using immunoprecipitation and confocal microscopy, we provide evidence that the actin nucleation-promoting factor cortactin is an SH3 domain-directed interaction partner of SLy2. Consistent with an important role of SLy2 for actin cytoskeletal reorganization, we further show that SLy2-transgenic B cells are severely defective in cell spreading. Together, our findings extend our mechanistic understanding of the immunoinhibitory roles of SLy2 in vivo and suggest that the physiological up-regulation of SLy2 observed upon B cell activation functions to counteract excessive B cell spreading.

Oligodendrocyte Myelin Glycoprotein Does Not Influence Node of Ranvier Structure or Assembly

Oligodendrocyte myelin glycoprotein (OMgp) is expressed by both neurons and oligodendrocytes in the CNS. It has been implicated in growth cone collapse and neurite outgrowth inhibition by signaling through the Nogo receptor and paired Ig-like receptor B (PirB). OMgp was also reported to be an extracellular matrix (ECM) protein surrounding CNS nodes of Ranvier and proposed to function as (1) an inhibitor of nodal collateral sprouting and (2) an important contributor to proper nodal and paranodal architecture. However, we show here that the anti-OMgp antiserum used in previous studies to define the functions of OMgp at nodes is not specific. Among all reported nodal ECM components, the antiserum exhibited strong cross-reactivity against versican V2 isoform, a chondroitin sulfate proteoglycan. Furthermore, the OMgp antiserum labeled OMgp-null nodes, but not nodes from versican V2-deficient mice, and preadsorption of the OMgp antiserum with recombinant versican V2 blocked nodal labeling. Analysis of CNS nodes in OMgp-null mice failed to reveal any nodal or paranodal defects, or increased nodal collateral sprouting, indicating that OMgp does not participate in CNS node of Ranvier assembly or maintenance. We successfully identified a highly specific anti-OMgp antibody and observed OMgp staining in white matter only after initiation of myelination. OMgp immunoreactivity decorated the surface of mature myelinated axons, but was excluded from compact myelin and nodes. Together, our results strongly argue against the nodal localization of OMgp and its proposed functions at nodes, and reveal OMgp's authentic localization relative to nodes and myelin.

Alleviation of Acute Graft-Verus-Host Disease Following Regulatory Dendritic Cell Treatment in Young and Older Mice.

Abstract 3550Poster Board III-487Older patients experience increased severity of graft-versus-host disease (GVHD) following allogeneic bone marrow transplant and host dendritic cells (DC) are a vital initiator of this disease.To evaluate the effect of age on GVHD severity and establish a model that can be used to study treatment of GVHD, a complete MHC mismatched bone marrow transplant (BMT) model was used to induce GVHD. Older (≥14 month) and young (3-4 month) mice succumbed to GVHD, but older mice experienced faster onset of weight loss and earlier mortality. Prophylactic treatment with syngeneic (host strain-derived) regulatory DC (DCreg) has been shown to decrease mortality of GHVD in young mice (Immunity 2003 18: 367-379). To determine whether this treatment also decreases the morbidity from GVHD in young mice and to evaluate its efficacy in older mice, day +2 BMT mice were treated with DCreg (hereafter referred to as DCreg treated BMT mice). As previously described, DCreg were generated by culture of bone marrow cells (BM) with IL-10, GM-CSF, and TGF-β1 (Immunity 2003 18: 367-379). All DCreg treated mice exhibited a brief period of GVHD and survived at least 45 days post-BMT; overall, older mice had increased morbidity and decreased long-term survival than young mice. In vivo analysis of regulatory and co-stimulatory DC molecules (PD-L1, PD-L2, CD103, CD40, CD80, and CD86) on host and donor-derived DC revealed no statistical differences between young and older DCreg treated BMT mice. Paired Ig-like receptor B (PIR B) is an inhibitory receptor expressed on DC and GVHD is exacerbated in PIR B−/− mice (Nat. Immunol. 2004 5:623-629). An antibody that recognizes both PIR A and PIR B was used to look at expression on DC in DCreg treated BMT mice. PIR A/B expression was increased in vivo on young host DC compared to older host DC. Transferred DCreg are host-strain derived and it is unclear if the PIR A/B expression in these experiments was on residual host DC or the transferred DCreg. Future utilization of green fluorescent protein positive (GFP+) DCreg will determine the origin of upregulated PIR A/B expression in vivo. In contrast to cultured stimulatory DC, young cultured DCreg revealed increased PIR A/B expression and were negative for co-stimulatory DC molecule expression. This difference in PIR A/B expression potentially provides an explanation for the disparate outcome of older DCreg treated BMT mice.Previous studies have implicated Treg in the induction of tolerance in DCreg treated mice (Immunity 2003 18: 367-379). However, the mechanism of tolerance maintenance has not been investigated. Abrogation of Treg mediated suppression in vivo with anti-GITR antibody (DTA-1) did not result in recurrence of GVHD or mortality in young mice, implying maintenance of the GVHD-free state is not Treg mediated in young mice.Human transplant recipients have a compromised immune system from both the transplant and immunosuppressive drug regimen that results in increased susceptibility to infection. Upon infection, GVHD recurrence often occurs. A murine influenza infection model was used to assess the donor immune system's capacity to respond to infection and remain free of GVHD in young DCreg treated BMT mice. Forty-five days or more post-BMT, once completely recovered from GVHD and demonstrated to be allo-reconstituted, the DCreg treated BMT mice were intra-nasally infected with 0.1LD50 of the H1N1 mouse adapted A/PR/8/34 influenza virus. DCreg treated BMT mice did not have a resurgence of GVHD following infection and displayed similar morbidity to control mice.In conclusion, DCreg treatment of BMT mice results in decreased morbidity and mortality from acute GVHD in both young and older mice relative to untreated BMT mice. However, the older mice still do not fully recover and exhibit increased morbidity relative to young mice. The maintenance of tolerance observed in young DCreg treated BMT mice does not appear to be Treg mediated. DCreg treated BMT mice experience similar morbidity and mortality as control mice during influenza infection and do not experience a resurgence of GVHD, implying an intact immune response to influenza infection.Future studies will specifically examine the requirement of PIR B on DCreg for successful inhibition of GVHD. Additional work will continue to investigate the mechanisms of induction and maintenance of tolerance in DCreg treated BMT mice.S.M.S. is supported by the National Institutes of Health (grant #5T32 AI07485). Disclosures:No relevant conflicts of interest to declare.

Ectopically expressed PIR-B on T cells constitutively binds to MHC class I and attenuates T helper type 1 responses

Activated mature T cells induce various inhibitory receptors implicated in maintaining peripheral tolerance in response to the trans-acting ligands. Interestingly, paired Ig-like receptor (PIR)-B, an inhibitory MHC class I receptor on B cells and myeloid cells, could be involved in regulating early T cell development because epitope for PIR is detected on pre-thymic T/NK progenitors but not on thymocytes or mature T cells. We hypothesized that PIR-B is not only a regulator for T cell development but is also detrimental if expressed on mature T cells. Here we demonstrated, using PIR-B-deficient fetuses, that PIR-B is indeed expressed on the T cell progenitors but failed to identify its distinctive roles in the development. Forced expression of PIR-B in thymocytes and mature T cells also resulted in no abnormalities in development. However, upon antigenic or allogeneic stimulation, peripheral T cells with the ectopic PIR-B showed reduced T(h) type 1 responses due to the suppression of proximal TCR signaling by constitutive binding of PIR-B to MHC class I on the same cell surface. Our findings suggest that T cell expression of PIR-B with the cis-interacting MHC class I is strictly prohibited in periphery so as to secure prompt immune responses.

Identification of LRRc17 as a Negative Regulator of Receptor Activator of NF-κB Ligand (RANKL)-induced Osteoclast Differentiation

Osteoblasts are the primary cells responsible for bone formation. They also support osteoclast formation from bone marrow precursors in response to osteotropic factors by inducing receptor activator of NF-kappaB ligand (RANKL) expression and down-regulating osteoprotegerin (OPG) production. In addition to the RANKL-RANK-OPG signaling axis, other factors produced by osteoblasts/stromal cells are involved in osteoclastogenesis. Here, we describe the identification and characterization of leucine-rich repeat-containing 17 (LRRc17), a member of the LRR superfamily that acts as a negative regulator of RANKL-induced osteoclast differentiation. Osteoblasts showed high levels of LRRc17 expression, which was down-regulated in response to the pro-osteoclastogenic factor 1,25-dihydroxyvitamin D(3). Recombinant LRRc17 protein inhibited RANKL-induced osteoclast differentiation from bone marrow precursors, whereas it did not affect the differentiation or activation of macrophages and dendritic cells. These results suggest that among the cell types derived from common myeloid precursors, LRRc17 specifically regulates osteoclasts. Further analysis revealed that LRRc17 attenuated RANKL-induced expression of NFATc1 by blocking phospholipase C-gamma signaling, which, in turn, inhibited RANKL-mediated osteoclast differentiation. Taken together, our results demonstrated a novel inhibitory activity of LRRc17 in RANKL-induced osteoclastogenesis.