TET1 and TDG Suppress Inflammatory Response in Intestinal Tumorigenesis: Implications for Colorectal Tumors With the CpG Island Methylator Phenotype.

BACKGROUND & AIMS: Aberrant DNA methylation is frequent in colorectal cancer (CRC), but underlying mechanisms and pathologic consequences are poorly understood. METHODS: We disrupted active DNA demethylation genes Tet1 and/or Tdg from ApcMin mice and characterized the methylome and transcriptome of colonic adenomas. Data were compared to human colonic adenocarcinomas (COAD) in The Cancer Genome Atlas. RESULTS: There were increased numbers of small intestinal adenomas in ApcMin mice expressing the TdgN151A allele, whereas Tet1-deficient and Tet1/TdgN151A-double heterozygous ApcMin colonic adenomas were larger with features of erosion and invasion. We detected reduction in global DNA hypomethylation in colonic adenomas from Tet1- and Tdg-mutant ApcMin mice and hypermethylation of CpG islands in Tet1-mutant ApcMin adenomas. Up-regulation of inflammatory, immune, and interferon response genes was present in Tet1- and Tdg-mutant colonic adenomas compared to control ApcMin adenomas. This up-regulation was also seen in murine colonic organoids and human CRC lines infected with lentiviruses expressing TET1 or TDG short hairpin RNA. A 127-gene inflammatory signature separated colonic adenocarcinomas into 4 groups, closely aligned with their microsatellite or chromosomal instability and characterized by different levels of DNA methylation and DNMT1 expression that anticorrelated with TET1 expression. Tumors with the CpG island methylator phenotype (CIMP) had concerted high DNMT1/low TET1 expression. TET1 or TDG knockdown in CRC lines enhanced killing by natural killer cells. CONCLUSIONS: Our findings reveal a novel epigenetic regulation, linked to the type of genomic instability, by which TET1/TDG-mediated DNA demethylation decreases methylation levels and inflammatory/interferon/immune responses. CIMP in CRC is triggered by an imbalance of methylating activities over demethylating activities. These mice represent a model of CIMP CRC.

The efficacy of combination treatment with elotuzumab and lenalidomide is dependent on crosstalk between natural killer cells, monocytes and myeloma cells.

Patients with refractory relapsed multiple myeloma respond to combination treatment with elotuzumab and lenalidomide. The mechanisms underlying this observation are not fully understood. Furthermore, biomarkers predictive of response have not been identified to date. To address these issues, we used a humanized myeloma mouse model and adoptive transfer of human natural killer (NK) cells to show that elotuzumab and lenalidomide treatment controlled myeloma growth, and this was mediated through CD16 on NK cells. In co-culture studies, we showed that peripheral blood mononuclear cells from a subset of patients with refractory relapsed multiple myeloma were effective killers of OPM2 myeloma cells when treated with elotuzumab and lenalidomide, and this was associated with significantly increased expression of CD54 on OPM2 cells. Furthermore, elotuzumab- and lenalidomide-induced OPM2 cell killing and increased OPM2 CD54 expression were dependent on both monocytes and NK cells, and these effects were not mediated by soluble factors alone. At the transcript level, elotuzumab and lenalidomide treatment significantly increased OPM2 myeloma cell expression of genes for trafficking and adhesion molecules, NK cell activation ligands and antigen presentation molecules. In conclusion, our findings suggest that multiple myeloma patients require elotuzumab- and lenalidomide-mediated upregulation of CD54 on autologous myeloma cells, in combination with NK cells and monocytes to mediate an effective anti-tumor response. Furthermore, our data suggest that increased myeloma cell CD54 expression levels could be a powerful predictive biomarker for response to elotuzumab and lenalidomide treatment.

The effect of repeated testing on judgement bias in domestic dogs (Canis familiaris).

Judgement bias paradigms are increasingly being used as a measure of affective state in dogs. Approach to an ambiguous stimulus is commonly used as a measure of affect, however, this may also be influenced by learning. This study directly measured the impact of learning on a commonly used judgement bias paradigm in the absence of an affective state manipulation. Dogs (N = 15) were tested on a judgement bias task across five sessions. The dogs' latency to approach a bowl placed in one of three ambiguous locations between non-baited (negative) and baited (positive) locations was measured. Results show that session number had a significant effect on the dogs' latencies to reach the ambiguous bowl locations, with post-hoc tests revealing that dogs were significantly slower to approach the locations as the number of sessions increased. Session number also had a significant effect on the number of times the dogs did not approach the bowl within 30 s of being released, with the number of no approaches generally increasing across sessions. When dog identity was included as a fixed effect, a significant effect on latency to approach was found, suggesting that some dogs were consistently faster than others across sessions. To assess whether the paradigm produced repeatable results, Intraclass Correlation Coefficients were used. A low degree of reliability was found between latencies to approach each bowl position across sessions. This study demonstrates that dogs learned that the ambiguous locations were not rewarded with repeated exposures, and that this impacted their responses. We conclude that this judgement bias paradigm may require further consideration if applied across multiple exposures and that repeated results should be interpreted with caution as they are likely impacted by learning.

Blocking the PCNA/NKp44 Checkpoint to Stimulate NK Cell Responses to Multiple Myeloma.

Multiple Myeloma (MM) is a devastating malignancy that evades immune destruction using multiple mechanisms. The NKp44 receptor interacts with PCNA (Proliferating Cell Nuclear Antigen) and may inhibit NK cells' functions. Here we studied in vitro the expression and function of PCNA on MM cells. First, we show that PCNA is present on the cell membrane of five out of six MM cell lines, using novel anti-PCNA mAb developed to recognize membrane-associated PCNA. Next, we stained primary bone marrow (BM) mononuclear cells from MM patients and showed significant staining of membrane-associated PCNA in the fraction of CD38+CD138+ BM cells that contain the MM cells. Importantly, blocking of the membrane PCNA on MM cells enhanced the activity of NK cells, including IFN-γ-secretion and degranulation. Our results highlight the possible blocking of the NKp44-PCNA immune checkpoint by the mAb 14-25-9 antibody to enhance NK cell responses against MM, providing a novel treatment option.

Impacts of pembrolizumab therapy on immune phenotype in patients with high-grade neuroendocrine neoplasms.

High grade neuroendocrine neoplasms (G3 NENs) are rare aggressive tumors with limited treatment options. Twenty-one previously treated patients with metastatic extra-pulmonary G3 NENs were treated with pembrolizumab. Baseline tumor samples were assessed for PD-L1 and tumor infiltrating lymphocytes (TIL). Peripheral blood samples drawn pre-treatment, prior to cycle three, and at disease progression were analyzed by flow cytometry. One patient achieved partial response, two had stable disease, and 18 exhibited progressive disease. The partially responding patient did not progress after 392 days, and the median progression-free survival (PFS) was 59 days. Longer PFS correlated independently with higher pre-treatment peripheral blood T-cell counts and lower pre-treatment activation state (CD69 expression) of naïve T cells and NK cells. Peripheral T-cell viability was reduced in patients with greater TILs. Post-treatment, T cells had reduced numbers of CD4+ cells, reduced PD-1 expression, increased activation of effector (CD62L-) cells, and increased expression of TIGIT. Baseline TIGIT expression on peripheral T cells also correlated positively with Ki67 in tumor. Patients with higher baseline T-cell expression of TIM-3 had shorter PFS. Despite limited activity of pembrolizumab, this study highlights the immune phenotype in this rare tumor type before and after treatment. High baseline peripheral T-cell count and reduced activation of T and NK cell subsets were associated with improved outcomes. Furthermore, increased post-treatment TIGIT and elevated baseline TIM-3 expression suggest that these may limit the efficacy of pembrolizumab, providing a rationale for combination immunotherapy (PD-1 with TIGIT and/or TIM-3 antibodies) to treat extra-pulmonary G3 NENs.

Pups Assisting Wellness for Staff (P.A.W.S.): Evaluating the impact of canine-assisted interventions on critical care staff wellness.

Acute care settings can expose staff to job-related stressors. Pups Assisting Wellness for Staff (P.A.W.S.) has been designed as an innovative approach to support acute care staff in the emergency department and intensive care unit. Initially, P.A.W.S. was implemented as a Comfort Dog Pilot and then expanded into a Therapy Dog Model. The Therapy Dog Model incorporated learnings from the Comfort Dog Pilot to evaluate the impact of the following: more dogs, using certified therapy dogs, and visiting at different times of the day. Results throughout the project demonstrated a positive impact on staff morale, staff stress, overall and staff satisfaction, and a strong desire for the project to continue. As a result and with a collaborative effort between a multidisciplinary team, P.A.W.S. has now been operationalized as a permanent program to support staff in acute care.

Immune phenotype of patients with stage IV metastatic inflammatory breast cancer.

BACKGROUND: Inflammatory breast cancer (IBC) is a rare but aggressive carcinoma characterized by severe erythema and edema of the breast, with many patients presenting in advanced metastatic disease. The "inflammatory" nature is not due to classic immune-mediated inflammation, but instead results from tumor-mediated blockage of dermal lymphatic ducts. Previous work has shown that expression of PD-L1 on tumor cells can suppress T cell activation in triple-negative (TN) non-IBC breast cancer. In the present work, we investigated immune parameters in peripheral blood of metastatic IBC patients to determine whether cellular components of the immune system are altered, thereby contributing to pathogenesis of the disease. These immune parameters were also compared to PD-1 and PD-L1 expression in IBC tumor biopsies. METHODS: Flow cytometry-based immune phenotyping was performed using fresh peripheral blood from 14 stage IV IBC patients and compared to 11 healthy age-similar control women. Immunohistochemistry for CD20, CD3, PD-1, and PD-L1 was performed on tumor biopsies of these metastatic IBC patients. RESULTS: IBC patients with Stage IV disease had lymphopenia with significant reductions in circulating T, B, and NK cells. Reductions were observed in all subsets of CD4+ T cells, whereas reductions in CD8+ T cells were more concentrated in memory subsets. Immature cytokine-producing CD56bright NK cells expressed higher levels of FcγRIIIa and cytolytic granule components, suggesting accelerated maturation to cytolytic CD56dim cells. Immunohistochemical analysis of tumor biopsies demonstrated moderate to high expression of PD-1 in 18.2% of patients and of PD-L1 in 36.4% of patients. Interestingly, a positive correlation was observed between co-expression levels of PD-L1 and PD-1 in tumor biopsies, and higher expression of PD-L1 in tumor biopsies correlated with higher expression of cytolytic granule components in blood CD4+ T cells and CD56dim NK cells, and higher numbers of CD8+ effector memory T cells in peripheral blood. PD-1 expression in tumor also correlated with increased infiltration of CD20+ B cells in the tumor. CONCLUSIONS: Our results suggest that while lymphocyte populations are severely compromised in stage IV IBC patients, an immune response toward the tumor had occurred in some patients, providing biological rationale to evaluate PD-1/PD-L1 immunotherapies for IBC.

Splice variants of human natural cytotoxicity receptors: novel innate immune checkpoints.

The natural cytotoxicity receptors (NCRs; NKp30, NKp44, and NKp46) were first defined as activating receptors on human NK cells that are important in recognition of and response to tumors. A flurry of recent research, however, has revealed that differential splicing can occur during transcription of each of the NCR genes, resulting in some transcripts that encode receptor isoforms with inhibitory functions. These alternative transcripts can arise in certain tissue microenvironments and appear to be induced by cytokines. Evidence indicates that some of the inhibitory NCRs are triggered by specific ligands, such as the interaction of the inhibitory isoform of NKp44 with PCNA on the surface of tumor cells. Here, we review the different NCR splice variants, cytokines that modulate their expression, their functional impacts on innate immune cells, and their differential expression in the contexts of cancer, pregnancy, and infections. The recent discovery of these inhibitory NCR isoforms has revealed novel innate immune checkpoints, many of which still lack defined ligands and clear mechanisms driving their expression. These NCR checkpoint pathways offer exciting potential therapeutic targets to manipulate innate immune functions under defined pathological conditions, such as cancer, pregnancy disorders, and pathogen exposure.

A Conserved HIV-1-Derived Peptide Presented by HLA-E Renders Infected T-cells Highly Susceptible to Attack by NKG2A/CD94-Bearing Natural Killer Cells.

Major histocompatibility class I (MHC-I)-specific inhibitory receptors on natural killer (NK) cells (iNKRs) tolerize mature NK cell responses toward normal cells. NK cells generate cytolytic responses to virus-infected or malignant target cells with altered or decreased MHC-I surface expression due to the loss of tolerizing ligands. The NKG2A/CD94 iNKR suppresses NK cell responses through recognition of the non-classical MHC-I, HLA-E. We used HIV-infected primary T-cells as targets in an in vitro cytolytic assay with autologous NK cells from healthy donors. In these experiments, primary NKG2A/CD94(+) NK cells surprisingly generated the most efficient responses toward HIV-infected T-cells, despite high HLA-E expression on the infected targets. Since certain MHC-I-presented peptides can alter recognition by iNKRs, we hypothesized that HIV-1-derived peptides presented by HLA-E on infected cells may block engagement with NKG2A/CD94, thereby engendering susceptibility to NKG2A/CD94(+) NK cells. We demonstrate that HLA-E is capable of presenting a highly conserved peptide from HIV-1 capsid (AISPRTLNA) that is not recognized by NKG2A/CD94. We further confirmed that HLA-C expressed on HIV-infected cells restricts attack by KIR2DL(+) CD56(dim) NK cells, in contrast to the efficient responses by CD56(bright) NK cells, which express predominantly NKG2A/CD94 and lack KIR2DLs. These findings are important since the use of NK cells was recently proposed to treat latently HIV-1-infected patients in combination with latency reversing agents. Our results provide a mechanistic basis to guide these future clinical studies, suggesting that ex vivo-expanded NKG2A/CD94(+) KIR2DL(-) NK cells may be uniquely beneficial.

Cutting Edge: Role of NK Cells and Surfactant Protein D in Dendritic Cell Lymph Node Homing: Effects of Ozone Exposure.

The roles of NK cells, surfactant protein D (SP-D), and IFN-γ, as well as the effect of ozone (O3) inhalation, were studied on recirculation of pulmonary dendritic cells (DC) to the mediastinal lymph nodes. O3 exposure and lack of SP-D reduced NK cell IFN-γ and lung tissue CCL21 mRNA expression and impaired DC homing to the mediastinal lymph nodes. Notably, addition of recombinant SP-D to naive mononuclear cells stimulated IFN-γ release in vitro. Because NKp46, a glycosylated membrane receptor, was necessary for dose-dependent SP-D binding to NK cells in vitro and DC migration in vivo, we speculate that SP-D may constitutively stimulate IFN-γ production by NK cells, possibly via NKp46. This mechanism could then initiate the IFN-γ/IL-12 feedback circuit, a key amplifier of DC lymph node homing. Inhibition of this process during an acute inflammatory response causes DC retention in the peripheral lung tissue and contributes to injury.

Central nervous system acute lymphoblastic leukemia: role of natural killer cells.

Central nervous system acute lymphoblastic leukemia (CNS-ALL) is a major clinical problem. Prophylactic therapy is neurotoxic, and a third of the relapses involve the CNS. Increased expression of interleukin 15 (IL-15) in leukemic blasts is associated with increased risk for CNS-ALL. Using in vivo models for CNS leukemia caused by mouse T-ALL and human xenografts of ALL cells, we demonstrate that expression of IL-15 in leukemic cells is associated with the activation of natural killer (NK) cells. This activation limits the outgrowth of leukemic cells in the periphery, but less in the CNS because NK cells are excluded from the CNS. Depletion of NK cells in NOD/SCID mice enabled combined systemic and CNS leukemia of human pre-B-ALL. The killing of human leukemia lymphoblasts by NK cells depended on the expression of the NKG2D receptor. Analysis of bone marrow (BM) diagnostic samples derived from children with subsequent CNS-ALL revealed a significantly high expression of the NKG2D and NKp44 receptors. We suggest that the CNS may be an immunologic sanctuary protected from NK-cell activity. CNS prophylactic therapy may thus be needed with emerging NK cell-based therapies against hematopoietic malignancies.

Going Beyond the 9-1-1 Call - What BC Emergency Health Services is Doing to Improve Timely Access to Emergency Care.

British Columbia Emergency Health Services (BCEHS) uses an internationally recognized Medical Priority Dispatch System to assign appropriate responses to 9-1-1 calls based on patients' clinical acuity. In 2015, 71% of Omega calls (classified as calls involving low acuity injuries) were assigned an ambulance. To better meet patients' needs, BCEHS collaborated with HealthLink BC's Nursing Services (HLBC NS) to audit over 2,000 calls. Based on the results, three Plan, Do, Study, Act (PDSA) cycles were implemented, yielding a 35% decrease in ambulances assigned and a 173% increase in referrals to HLBC NS to provide more suitable support. Ultimately, the interventions allowed these ambulances to be reallocated to more critical patients.

Integrins Influence the Size and Dynamics of Signaling Microclusters in a Pyk2-dependent Manner.

Integrin engagement on lymphocytes initiates "outside-in" signaling that is required for cytoskeleton remodeling and the formation of the synaptic interface. However, the mechanism by which the "outside-in" signal contributes to receptor-mediated intracellular signaling that regulates the kinetics of granule delivery and efficiency of cytolytic activity is not well understood. We have found that variations in ICAM-1 expression on tumor cells influence killing kinetics of these cells by CD16.NK-92 cytolytic effectors suggesting that changes in integrin ligation on the effector cells regulate the kinetics of cytolytic activity by the effector cells. To understand how variations of the integrin receptor ligation may alter cytolytic activity of CD16.NK-92 cells, we analyzed molecular events at the contact area of these cells exposed to planar lipid bilayers that display integrin ligands at different densities and activating CD16-specific antibodies. Changes in the extent of integrin ligation on CD16.NK-92 cells at the cell/bilayer interface revealed that the integrin signal influences the size and the dynamics of activating receptor microclusters in a Pyk2-dependent manner. Integrin-mediated changes of the intracellular signaling significantly affected the kinetics of degranulation of CD16.NK-92 cells providing evidence that integrins regulate the rate of target cell destruction in antibody-dependent cell cytotoxicity (ADCC).

Regulation of natural cytotoxicity receptors by heparan sulfate proteoglycans in -cis: A lesson from NKp44.

NKp44 (NCR2) is a distinct member of natural cytotoxicity receptors (NCRs) family that can induce cytokine production and cytolytic activity in human NK cells. Heparan sulfate proteoglycans (HSPGs) are differentially expressed in various normal and cancerous tissues. HSPGs were reported to serve as ligands/co-ligands for NKp44 and other NCRs. However, HSPG expression is not restricted to either group and can be found also in NK cells. Our current study reveals that NKp44 function can be modulated through interactions with HSPGs on NK cells themselves in -cis rather than on target cells in -trans. The intimate interaction of NKp44 and the NK cell-associated HSPG syndecan-4 (SDC4) in -cis can directly regulate membrane distribution of NKp44 and constitutively dampens the triggering of the receptor. We further demonstrate, that the disruption of NKp44 and SDC4 interaction releases the receptor to engage with its ligands in -trans and therefore enhances NKp44 activation potential and NK cell functional response.

The ap-2 clathrin adaptor mediates endocytosis of an inhibitory killer cell Ig-like receptor in human NK cells.

Stable surface expression of human inhibitory killer cell Ig-like receptors (KIRs) is critical for controlling NK cell function and maintaining NK cell tolerance toward normal MHC class I(+) cells. Our recent experiments, however, have found that Ab-bound KIR3DL1 (3DL1) readily leaves the cell surface and undergoes endocytosis to early/recycling endosomes and subsequently to late endosomes. We found that 3DL1 internalization is at least partially mediated by an interaction between the µ2 subunit of the AP-2 clathrin adaptor complex and ITIM tyrosine residues in the cytoplasmic domain of 3DL1. Disruption of the 3DL1/µ2 interaction, either by mutation of the ITIM tyrosines in 3DL1 or mutation of µ2, significantly diminished endocytosis and increased surface expression of 3DL1 in human primary NK cells and cell lines. Furthermore, we found that the 3DL1/AP-2 interaction is diminished upon Ab engagement with the receptor, as compared with untreated cells. Thus, we have identified AP-2-mediated endocytosis as a mechanism regulating the surface levels of inhibitory KIRs through their ITIM domains. Based on our results, we propose a model in which nonengaged KIRs are internalized by this mechanism, whereas engagement with MHC class I ligand would diminish AP-2 binding, thereby prolonging stable receptor surface expression and promoting inhibitory function. Furthermore, this ITIM-mediated mechanism may similarly regulate the surface expression of other inhibitory immune receptors.

Integrin receptors on tumor cells facilitate NK cell-mediated antibody-dependent cytotoxicity.

NK cells that mediate ADCC play an important role in tumor-specific immunity. We have examined factors limiting specific lysis of tumor cells by CD16.NK-92 cells induced by CNTO 95LF antibodies recognizing αV integrins that are overexpressed on many tumor cells. Although all tested tumor cells were killed by CD16.NK-92 effectors in the presence of the antibodies, the killing of target cells with a low level of ICAM-1 expression revealed a dramatic decrease in their specific lysis at high antibody concentration, revealing a dose limiting effect. A similar effect was also observed with primary human NK cells. The effect was erased after IFN-γ treatment of tumor cells resulting in upregulation of ICAM-1. Furthermore, killing of the same tumor cells induced by Herceptin antibody was significantly impaired in the presence of CNTO 95Ala-Ala antibody variant that blocks αV integrins but is incapable of binding to CD16. These data suggest that αV integrins on tumor cells could compensate for the loss of ICAM-1 molecules, thereby facilitating ADCC by NK cells. Thus, NK cells could exercise cytolytic activity against ICAM-1 deficient tumor cells in the absence of proinflammatory cytokines, emphasizing the importance of NK cells in tumor-specific immunity at early stages of cancer.

Genome-wide siRNA screen reveals a new cellular partner of NK cell receptor KIR2DL4: heparan sulfate directly modulates KIR2DL4-mediated responses.

KIR2DL4 (CD158d) is a distinct member of the killer cell Ig-like receptor (KIR) family in human NK cells that can induce cytokine production and cytolytic activity in resting NK cells. Soluble HLA-G, normally expressed only by fetal-derived trophoblast cells, was reported to be a ligand for KIR2DL4; however, KIR2DL4 expression is not restricted to the placenta and can be found in CD56(high) subset of peripheral blood NK cells. We demonstrated that KIR2DL4 can interact with alternative ligand(s), expressed by cells of epithelial or fibroblast origin. A genome-wide high-throughput siRNA screen revealed that KIR2DL4 recognition of cell-surface ligand(s) is directly regulated by heparan sulfate (HS) glucosamine 3-O-sulfotransferase 3B1 (HS3ST3B1). KIR2DL4 was found to directly interact with HS/heparin, and the D0 domain of KIR2DL4 was essential for this interaction. Accordingly, exogenous HS/heparin can regulate cytokine production by KIR2DL4-expressing NK cells and HEK293T cells (HEK293T-2DL4), and induces differential localization of KIR2DL4 to rab5(+) and rab7(+) endosomes, thus leading to downregulation of cytokine production and degradation of the receptor. Furthermore, we showed that intimate interaction of syndecan-4 (SDC4) HS proteoglycan (HSPG) and KIR2DL4 directly affects receptor endocytosis and membrane trafficking.

Prohibitins and the cytoplasmic domain of CD86 cooperate to mediate CD86 signaling in B lymphocytes.

CD86 engagement on a CD40L/IL-4-primed murine B cell activates signaling intermediates that promote NF-κB activation to increase Oct-2 and mature IgG1 mRNA and protein expression, as well as the rate of IgG1 transcription, without affecting class switch recombination. One of the most proximal signaling intermediates identified is phospholipase Cγ2, a protein reported to bind tyrosine residues, which are absent in the cytoplasmic domain of CD86. Using a proteomics-based identification approach, we show that the tyrosine-containing transmembrane adaptor proteins prohibitin (Phb)1 and Phb2 bind to CD86. The basal expression of Phb1/2 and association with CD86 was low in resting B cells, whereas the level of expression and association increased primarily after priming with CD40. The CD86-induced increase in Oct-2 and IgG1 was less when either Phb1/2 expression was reduced by short hairpin RNA or the cytoplasmic domain of CD86 was truncated or mutated at serine/threonine protein kinase C phosphorylation sites, which did not affect Phb1/2 binding to CD86. Using this approach, we also show that Phb1/2 and the CD86 cytoplasmic domain are required for the CD86-induced phosphorylation of IκBα, which we previously reported leads to NF-κB p50/p65 activation, whereas only Phb1/2 was required for the CD86-induced phosphorylation of phospholipase Cγ2 and protein kinase Cα/ß(II), which we have previously reported leads to NF-κB (p65) phosphorylation and subsequent nuclear translocation. Taken together, these findings suggest that Phb1/2 and the CD86 cytoplasmic domain cooperate to mediate CD86 signaling in a B cell through differential phosphorylation of distal signaling intermediates required to increase IgG1.

B-cell adaptor for PI3K (BCAP) negatively regulates Toll-like receptor signaling through activation of PI3K.

Toll-like receptors (TLRs) recognize pathogens and their components, thereby initiating immune responses to infectious organisms. TLR ligation leads to the activation of NF-κB and MAPKs through well-defined pathways, but it has remained unclear how TLR signaling activates PI3K, which provides an inhibitory pathway limiting TLR responses. Here, we show that the signaling adapter B-cell adaptor for PI3K (BCAP) links TLR signaling to PI3K activation. BCAP-deficient macrophages and mice are hyperresponsive to TLR agonists and have reduced PI3K activation. The ability of BCAP to inhibit TLR responses requires its capacity to bind PI3K. BCAP is constitutively phosphorylated and associated with the p85 subunit of PI3K in macrophages. This tyrosine-phosphorylated BCAP is transiently enriched in the membrane fraction in response to LPS treatment, suggesting a model whereby TLR signaling causes the phosphorylation of the small amount of BCAP that is associated with membranes in the resting state or the translocation of phosphorylated BCAP from the cytoplasm to the membrane. This accumulation of tyrosine-phosphorylated BCAP at the membrane with its associated PI3K would then allow for the catalysis of Ptd Ins P2 to Ptd Ins P3 and downstream PI3K-dependent signals. Therefore, BCAP is an essential activator of the PI3K pathway downstream of TLR signaling, providing a brake to limit potentially pathogenic excessive TLR responses.