ITAM Signaling Research Articles

Epstein-Barr Virus Latent Membrane Protein-2A Induces ITAM/Syk- and Akt-Dependent Epithelial Migration through αV-Integrin Membrane Translocation

Epstein-Barr virus (EBV) is a highly prevalent herpesvirus associated with epithelial cancers, including nasopharyngeal carcinoma (NPC). The EBV protein latent membrane protein 2 (LMP2) is expressed in NPC tumor tissue and has been shown to induce transformation, inhibit differentiation, and promote migration of epithelial cells. In this study, the effect of LMP2A on migration of human epithelial cells was further analyzed. LMP2A expression induced migration in human foreskin keratinocytes (HFK) and HaCaT keratinocytes measured by wound healing scratch assay and chemoattractant-induced Transwell migration assay. The induction of migration by LMP2A required the ITAM signaling domain of LMP2A and activation of the Syk tyrosine kinase. LMP2A-induced Transwell migration required the Akt signaling pathway, and activation of Akt by LMP2A required the ITAM signaling domain of LMP2A. LMP2A also induced phosphorylation of the Akt target GSK3β, a Wnt signaling mediator that has been shown to regulate the activity of focal adhesion kinase (FAK), a tyrosine kinase activated by clustering and ligand interaction of integrins. Inhibition of either FAK or its signaling mediator Src kinase inhibited LMP2A-induced migration. Interestingly, αV-integrin was greatly increased in membrane-enriched fractions by LMP2A, and a neutralizing antibody to αV-integrin blocked migration, suggesting that the effects of LMP2A on membrane-localized αV-integrin promoted migration. The results of this study indicate that LMP2A expression in human epithelial cells induces αV-integrin-dependent migration through a mechanism requiring ITAM-mediated Syk and Akt activation and inducing membrane translocation or stabilization of αV-integrin and FAK activation. The specific effects of LMP2A on an integrin with a diverse repertoire of ligand specificities could promote migration of different cell types and be initiated by multiple chemoattractants.

CD2AP/SHIP1 Complex Positively Regulates Plasmacytoid Dendritic Cell Receptor Signaling by Inhibiting the E3 Ubiquitin Ligase Cbl

The human plasmacytoid dendritic cell (pDC) receptor BDCA2 forms a complex with the adaptor FcεR1γ to activate an ITAM-signaling cascade. BDCA2 receptor signaling negatively regulates the TLR7/9-mediated type 1 IFN responses in pDCs, which may play a key role in controlling self-DNA/RNA-induced autoimmunity. We report in this article that CD2-associated adaptor protein (CD2AP), which is highly expressed in human pDCs, positively regulates BDCA2/FcεR1γ receptor signaling. By immunoprecipitation and mass spectrometry analyses, we found that CD2AP bound to SHIP1. Knockdown of CD2AP or SHIP1 reduced the BDCA2/FcεR1γ-mediated ITAM signaling and blocked its inhibition of TLR9-mediated type 1 IFN production. Knockdown of CD2AP or SHIP1 also enhanced the ubiquitination and degradation of Syk and FcεR1γ that was mediated by the E3 ubiquitin ligase Cbl. This led us to discover that, upon BDCA2 cross-linking, the CD2AP/SHIP1 complex associated with Cbl and inhibited its E3 ubiquitin ligase activity. In human primary pDCs, cross-linking of the BDCA2/FcεR1γ complex induced the recruitment of the CD2AP/SHIP1/Cbl complex to the plasma membrane of pDCs, where it colocalized with the BDCA2/FcεR1γ complex. Therefore, CD2AP positively regulates BDCA2/FcεR1γ signaling by forming a complex with SHIP1 to inhibit the E3 ubiquitin ligase Cbl.

ITAM-Coupled Receptors Inhibit IFNAR Signaling and Alter Macrophage Responses to TLR4 and Listeria mono cytogenes

ITAM-coupled receptors play an essential role in regulating macrophage activation and function by cross-regulating signaling from heterologous receptors. We investigated mechanisms by which ITAM-associated receptors inhibit type I IFN (IFN-α/β) signaling in primary human macrophages and tested the effects of simultaneous ligation of ITAM-associated receptors and TLR4 on TLR4-induced Jak-STAT signaling that is mediated by autocrine IFN-β. Preligation of ITAM-coupled β2 integrins and FcγRs inhibited proximal signaling by the type I IFN receptor IFNAR. Cross-inhibition of IFNAR signaling by β2 integrins resulted in decreased Jak1 activation and was mediated by partial downregulation of the IFNAR1 subunit and MAPK-dependent induction of USP18, which blocks the association of Jak1 with IFNAR2. Simultaneous engagement of ITAM-coupled β2 integrins or Dectin-1 with TLR4 did not affect TLR4-induced direct activation of inflammatory target genes such as TNF or IL6 but abrogated subsequent induction of IFN response genes that is mediated by autocrine IFN-β signaling. Type I IFNs promote macrophage death postinfection by Listeria monocytogenes. Consequently, attenuation of IFN responses by β2 integrins protected primary human macrophages from L. monocytogenes-induced apoptosis. These results provide a mechanism for cross-inhibition of type I IFN signaling by ITAM-coupled β2 integrins and demonstrate that ITAM signaling qualitatively modulates macrophage responses to pathogen-associated molecular patterns and pathogens by selectively suppressing IFN responses.

An Important Role for Rac1 in Glycoprotein Ib-IX Induced Platelet Activation,

Abstract 3250The platelet receptor for von Willebrand factor, the glycoprotein Ib-IX (GPIb-IX) complex, not only plays a critical role in mediating platelet adhesion at sites of vascular injury, but also transmits signals leading to platelet activation. GPIb-IX mediated activation of the ligand binding function of integrin αIIbβ3 is required for stable platelet adhesion under high shear rate conditions. Previous studies have suggested that ligand occupancy of GPIb-IX leads to activation of the Src family kinase Lyn, phosphoinositide 3-kinase (PI3K), Akt, cGMP-dependent protein kinase, and the MAPK and ITAM signaling pathways. However, it remains unclear how the PI3K/Akt pathway is activated during GPIb-IX dependent signal transduction. Using platelet-specific conditional Rac1−/− mice and the Rac1 specific inhibitor NSC23766 (NSC), we examined the role of Rac1 in GPIb-IX induced platelet activation. Platelet-specific conditional Rac1−/− mice were generated by crossing Pf4-Cre transgenic mice with mice containing the Rac1 conditional allele. Rac1−/− mouse platelets had a defect in VWF/botrocetin induced aggregation, TXA2 synthesis, and ATP secretion. Rac1−/− mouse platelets were also defective in stable adhesion to VWF under shear stress and in spreading on immobilized VWF, as compared to WT. The defects observed in Rac1−/− mouse platelets were recapitulated in human platelets treated with the Rac1 inhibitor NSC23766. Importantly, we show that ligand binding to GPIb-IX induced Rac1 activation, which was abolished in Lyn−/− mouse platelets. We also show that Rac1−/− (or NSC-treated) platelets have a defect in GPIb-IX induced Akt and P38 MAPK activation. Altogether, this study reveals that Rac1 plays an important role in GPIb-IX dependent platelet activation and stable adhesion to VWF under shear stress. Rac1 is activated downstream of Lyn and is important for GPIb-IX and Lyn-dependent activation of Akt and MAPK signaling pathways. Disclosures:No relevant conflicts of interest to declare.

Ly49D-Mediated ITAM Signaling in Immature Thymocytes Impairs Development by Bypassing the Pre-TCR Checkpoint

Activating and inhibitory NK receptors regulate the development and effector functions of NK cells via their ITAM and ITIM motifs, which recruit protein tyrosine kinases and phosphatases, respectively. In the T cell lineage, inhibitory Ly49 receptors are expressed by a subset of activated T cells and by CD1d-restricted NKT cells, but virtually no expression of activating Ly49 receptors is observed. Using mice transgenic for the activating receptor Ly49D and its associated ITAM signaling DAP12 chain, we show in this article that Ly49D-mediated ITAM signaling in immature thymocytes impairs development due to a block in maturation from the double negative (DN) to double positive (DP) stages. A large proportion of Ly49D/DAP12 transgenic thymocytes were able to bypass the pre-TCR checkpoint at the DN3 stage, leading to the appearance of unusual populations of DN4 and DP cells that lacked expression of intracellular (ic) TCRβ-chain. High levels of CD5 were expressed on ic TCRβ(-) DN and DP thymocytes from Ly49D/DAP12 transgenic mice, further suggesting that Ly49D-mediated ITAM signaling mimics physiological ITAM signaling via the pre-TCR. We also observed unusual ic TCRβ(-) single positive thymocytes with an immature CD24(high) phenotype that were not found in the periphery. Importantly, thymocyte development was completely rescued by expression of an Ly49A transgene in Ly49D/DAP12 transgenic mice, indicating that Ly49A-mediated ITIM signaling can fully counteract ITAM signaling via Ly49D/DAP12. Collectively, our data indicate that inappropriate ITAM signaling by activating NK receptors on immature thymocytes can subvert T cell development by bypassing the pre-TCR checkpoint.

Inhibitory ITAM Signaling Traps Activating Receptors with the Phosphatase SHP-1 to Form Polarized “Inhibisome” Clusters

The ability of immunoreceptor tyrosine-based activation motif (ITAM)-bearing receptors to inhibit, rather than activate, signaling by other receptors is a regulatory mechanism of immune homeostasis. However, it remains unclear how inhibitory ITAM (ITAMi) receptor signaling and Src homology 2 (SH2) domain-containing phosphatase-1 (SHP-1), which is recruited to ITAMs, target multiple heterologous activating responses without coaggregating with the associated activating receptors. We found that ITAMi signaling triggered by the binding of monomeric ligands to the type I immunoglobulin A (IgA) Fc receptor (FcαRI) induced its dynamic cosegregation with heterologous activating receptors, signaling effectors, and the inhibitory phosphatase SHP-1 into polarized intracellular clusters that we call "inhibisomes." Formation of inhibisomes was preceded by the recruitment of FcαRI and SHP-1 into lipid rafts. Cosegregation required the depolymerization of actin, which depended on SHP-1, and inhibisome formation was abolished by knockdown of SHP-1 and by actin-depolymerizing drugs. Thus, SHP-1- and actin depolymerization-dependent spatiotemporal compartmentalization of ITAMi-containing receptors into lipid rafts, regions associated with intracellular signaling, represents a key event in the integration of ITAMi-mediated inhibitory signals.

Impact of TLR9 on hyper-responsive ITAM signaling in a mouse model of SLE (47.24)

Abstract Anti-nuclear autoantibody (ANA) production by autoreactive B cells is a hallmark symptom of the autoimmune disease Systemic Lupus Erythematosus (SLE), which has a complex genetic etiology with environmental influences. In the CD45E613R knock-in mouse model of SLE, mice homozygous for an activating point mutation in CD45 develop high titer anti-dsDNA autoantibodies on the BALB/c (BA) and not the C57BL6/J (B6) or 129/Sv genetic backgrounds despite hyper-responsive ITAM signaling in several immune lineages in each genetic background. A screen for genetic modifiers of autoantibody production identified the candidate gene Tlr9. Sequence analysis of B6 and BA alleles of Tlr9 indicate several point mutations, resulting in five amino acid substitutions in TLR9. Splenic CD11b+ myeloid cells from BA express lower levels of TLR9 and BA-derived BMDCs are less responsive to synthetic TLR9 ligand CpG DNA. BA B cells are also less responsive to TLR9-mediated signaling than B6 B cells despite similar levels of TLR9 protein, indicating the BA TLR9 allele is hypo-responsive. Genetic ablation of TLR9 in CD45E613R mice on the non-autoreactive B6 genetic background augments disease, with mice developing splenomegaly and increased frequencies of memory T cells by 3 months of age. Some aged animals also developed ANAs, supporting the hypothesis that reduced TLR9 responsiveness permits increased autoreactivity in the context of hyper-responsive ITAM signaling.

PTPN4 and PTPN3 regulation of ITAM containing proteins (50.9)

Abstract The T cell receptor (TCR) uses a highly conserved ITAM signaling pathway to transduce intracellular signals following appropriate ligand binding. Activations of the ITAM signaling pathway is tightly controlled by diverse families of protein tyrosine kinases and protein tyrosine phosphatases. In earlier studies, we reported that two PTPases (PTPN3 and PTPN4) could directly dephosphorylate the tyrosine residues in the CD3 z ITAMs. However, T cells from both PTPN3 and PTPN4-deficient mice exhibit normal CD3 regulated ITAM signaling pathways. Since PTPN3 and PTPN4 have FERM domains that localize these enzymes to the cytoskeleton, it is possible that these PTPases target distinct proteins. Interestingly, moesin, ezrin, and dectin-1 are three cytoskeletal associated proteins that use ITAM sequences to transduce intracellular signals. To examine whether these distinct ITAM-containing proteins were dephosphorylated by PTPN3 or PTPN4, a combination of transient transfection assays and cell signaling studies in PTPase-deficient dendritic cells were performed. These experiments will reveal whether this PTPase-family dephosphorylates distinct ITAM-containing proteins.

A unique domain in RANK is required for Gab2 and PLCγ2 binding to establish osteoclastogenic signals

TRAF6 is essential for osteoclastogenesis and for both RANK- and CD40-mediated activation of IKK and MAPKs. RANK, but not CD40, can promote osteoclastogenesis because only RANK induces NFATc1 activation through PLCgamma2-induced Ca(2+) oscillations together with the co-stimulatory signals emanating from immune receptors linked to ITAM-containing adaptors. These previous data suggest that RANK harbors a unique domain that functions in concert with the TRAF6-binding site in osteoclastogenesis. Here we identify such a domain, highly conserved domain in RANK (HCR), which is dispensable for the early phase of RANK and ITAM signaling but is essential for their late-phase signaling, including sustained activation of NF-kappaB and PLCgamma2 leading to NFATc1 activation. HCR recruits an adaptor protein, Gab2, which further associates with PLCgamma2 in the late phase. Formation of the HCR-mediated signaling complex could account for the sustained activation of NF-kappaB and PLCgamma2. The present study identifies HCR as a unique domain that plays a critical role in the long-term linkage between RANK and ITAM signals, providing a molecular basis for therapeutic strategies.

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.

Cutting Edge: Rescue of Pre-TCR but Not Mature TCR Signaling in Mice Expressing Membrane-Targeted SLP-76

SLP-76 (Src homology 2 domain-containing leukocyte phosphoprotein of 76 kDa) organizes signaling from immunoreceptors, including the platelet collagen receptor, the pre-TCR, and the TCR, and is required for T cell development. In this study we examine a mouse in which wild-type SLP-76 is replaced with a mutant constitutively targeted to the cell membrane. Membrane-targeted SLP-76 (MTS) supports ITAM signaling in platelets and from the pre-TCR. Signaling from the mature TCR, however, is defective in MTS thymocytes, resulting in failed T cell differentiation. Defective thymic selection by MTS is not rescued by a SLP-76 mutant whose localization is restricted to the cytosol. Thus, fixed localization of SLP-76 reveals differential requirements for the subcellular localization of signaling complexes downstream of the pre-TCR vs mature TCR.

A full complement of CD3 ζ ITAMs is necessary for NKT cell development and function (85.11)

Abstract Invariant natural killer T (iNKT) cells express a canonical αβ TCR that recognizes glycolipids embedded in non-classical MHC molecules. These cells develop following TCR interactions with CD1d/glycolipid complexes expressed on thymocytes. The αβ TCR is non-covalently associated with six signaling proteins, CD3 ζζ and CD3 γε/δε, that signal through ITAMs. It is unknown whether a full complement of ITAMs is required for iNKT cell development and function. We previously generated CD3 ζ transgenic mice with mutations in the first (YF1,2) or all three CD3 ζ ITAMs (YF1-6) to reduce ITAM signaling. iNKT cell development was analyzed in these mice. Compared to C57BL/6 controls (0.43%; 1.04%), the percentage and absolute numbers of iNKT cells were significantly reduced in the thymus and spleen of YF1,2 (0.008%; 0.023%; 11- and 19-fold) and YF1-6 (0.044%; 0.11%, 50- and 100-fold) mice. Comparative phenotyping suggested that the iNKT cells had an early developmental block in the YF1,2 and YF1-6 mice as evidenced by an increased percentage of iNKT cells expressing CD24 compared to C57BL/6 mice. These data indicate that a full complement of CD3 ζ ITAMs is required for proper iNKT cell development. We are currently assessing the functionality of iNKT cells in the various mice in response to glycolipids or following infections with the pathogenic bacteria Borrelia burgdorferi .

Ostéoimmunologie : une vision globale et intégrée du tissu squelettique et du système immunitaire

Study of dysregulations of the immune system resulting in abnormal bone homeostasis has led to the emergence of the field of osteoimmunology. Among the interactions between the immune system and bone, several signaling molecules and pathways previously identified in immune cells have been shown to be key players in the osteoclast, the bone resorbing cell. Osteoclast differentiation requires two signaling pathways: the RANK/RANKL pathway and the signal initiated by the ITAM-harboring adaptor proteins DAP12 and FcRgamma. Until recently, it was unclear how RANK and ITAM signals merge to cooperatively stimulate activation of NFATc1, the master transcription factor in osteoclastogenesis. A recent study from H. Takayanagi's group has shown that the tyrosine kinases Btk and Tec form a multiprotein complex with adaptor molecules such as BLNK, that is able to integrate these two signaling pathways and thus stimulate osteoclastogenesis. Taken together, these new data open novel clinical perspectives especially for osteoarticular inflammatory diseases.

ITAM Signaling by Vav Family Rho Guanine Nucleotide Exchange Factors Regulates Interstitial Transit Rates of Neutrophils In Vivo

BackgroundIn response to infection, neutrophils are quickly recruited from the blood into inflamed tissues. The interstitial migration of neutrophils is crucial for the efficient capture and control of rapidly proliferating microbes before microbial growth can overwhelm the host's defenses. However, the molecular mechanisms that regulate interstitial migration are incompletely understood.Methodology/Principal FindingsHere, we use two-photon microscopy (2PM) to study discrete steps of neutrophil responses during subcutaneous infection with bacteria. Our study demonstrates that signals emanating from ITAM-containing receptors mediated by Vav family Rho GEFs control the velocity, but not the directionality, of neutrophil migration towards sites of bacterial infection.Conclusions/SignificanceHere we show that during neutrophil migration towards sites of bacterial infection, signals emanating from ITAM-containing receptors specifically control interstitial neutrophil velocity.

Ly49D Engagement on T Lymphocytes Induces TCR-Independent Activation and CD8 Effector Functions That Control Tumor Growth

Recent data showing expression of activating NK receptors (NKR) by conventional T lymphocytes raise the question of their role in the triggering of TCR-independent responses that could be damaging for the host. Transgenic mice expressing the activating receptor Ly49D/DAP12 offer the opportunity to better understand the relevance of ITAM signaling in the biology of T cells. In vitro experiments showed that Ly49D engagement on T lymphocytes by a cognate MHC class I ligand expressed by Chinese hamster ovary (CHO) cells or by specific Ab triggered cellular activation of both CD4 and CD8 populations with modulation of activation markers and cytokine production. The forced expression of the ITAM signaling chain DAP12 is mandatory for Ly49D-transgenic T cell activation. In addition, Ly49D stimulation induced T lymphocyte proliferation, which was much stronger for CD8 T cells. Phenotypic analysis of anti-Ly49D-stimulated CD8 T cells and their ability to produce high levels of IFN-gamma and to kill target cells indicate that Ly49D ligation generates effector cytotoxic CD8 T cells. Ly49D engagement by itself also triggered cytotoxic activity of activated CD8 T cells. Adoptive transfer experiments confirmed that Ly49D-transgenic CD8 T cells are able to control growth of CHO tumor cells or RMA cells transfected with Hm1-C4, the Ly49D ligand normally expressed by CHO. In conclusion, Ly49D engagement on T cells leads to T cell activation and to a full range of TCR-independent effector functions of CD8 T cells.

“Dangerous Crystals”

Uric acid (UA) crystals are a potent stimulator of inflammation, but how they activate immune cells is not known. In this issue of Immunity, Ng et al. (2008) provide evidence suggesting that UA activates the Syk kinase via membrane cholesterol.

Requirements of SLP76 tyrosines in ITAM and integrin receptor signaling and in platelet function in vivo

Src homology 2 domain–containing leukocyte phosphoprotein of 76 kD (SLP76), an adaptor that plays a critical role in platelet activation in vitro, contains three N-terminal tyrosine residues that are essential for its function. We demonstrate that mice containing complementary tyrosine to phenylalanine mutations in Y145 (Y145F) and Y112 and Y128 (Y112/128F) differentially regulate integrin and collagen receptor signaling. We show that mutation of Y145 leads to severe impairment of glycoprotein VI (GPVI)–mediated responses while preserving outside-in integrin signaling. Platelets from Y112/128F mice, although having mild defects in GPVI signaling, exhibit defective actin reorganization after GPVI or αIIbβ3 engagement. The in vivo consequences of these signaling defects correlate with the mild protection from thrombosis seen in Y112/128F mice and the near complete protection observed in Y145F mice. Using genetic complementation, we further demonstrate that all three phosphorylatable tyrosines are required within the same SLP76 molecule to support platelet activation by GPVI.

Tyrosine Kinases Btk and Tec Regulate Osteoclast Differentiation by Linking RANK and ITAM Signals

Certain autoimmune diseases result in abnormal bone homeostasis, but association of immunodeficiency with bone is poorly understood. Osteoclasts, which derive from bone marrow cells, are under the control of the immune system. Differentiation of osteoclasts is mainly regulated by signaling pathways activated by RANK and immune receptors linked to ITAM-harboring adaptors. However, it is unclear how the two signals merge to cooperate in osteoclast differentiation. Here we report that mice lacking the tyrosine kinases Btk and Tec show severe osteopetrosis caused by a defect in bone resorption. RANK and ITAM signaling results in formation of a Btk(Tec)/BLNK(SLP-76)-containing complex and PLCgamma-mediated activation of an essential calcium signal. Furthermore, Tec kinase inhibition reduces osteoclastic bone resorption in models of osteoporosis and inflammation-induced bone destruction. Thus, this study reveals the importance of the osteoclastogenic signaling complex composed of tyrosine kinases, which may provide the molecular basis for a new therapeutic strategy.

The CD3 ζ Subunit of the T Cell Receptor is Required for an Efficient Immune Response During Bacterial Infection

The T cell receptor (TCR) complex is comprised of a ligand binding αβ heterodimer and two signaling modules, CD3 ζζ and CD3 γε/δε. Each signaling subunit has at least one copy of an ITAM signaling motif. TCR engagement results in bi‐phosphorylation of the ITAM on tyrosine residues, allowing ZAP‐70 to complex. This initiates an intracellular signaling cascade, leading to T cell activation and effector function.We generated CD3 ζ transgenic mice (YF1‐6) lacking all forms of phospho‐ζ. Previous data have shown that phospho‐ζ is not required for T cell development or signaling. Conversely, after TCR stimulation, CD8+ T cells from YF1‐6 mice produce 2‐fold less IFN‐γ than C57BL/6 mice. To determine the impact of this difference, we established a Listeria monocytogenes (LM) infection model. We found that YF1‐6 mice could not clear primary LM infections and had 5 to 50‐fold higher CFUs in the liver and spleen compared to control mice. Yet, after secondary infection, YF1‐6 and C57BL/6 mice cleared the bacteria. This suggests that phospho‐ζ is required for innate cell function during LM infection. We are examining whether this results from aberrant CD8+ T or NK cell function, as both populations are thought to be important for early IFN‐γ production and LM clearance. Defining the role of phospho‐ζ during innate immune responses to infections may reveal new therapeutic targets. Supported by NIH T32 AI005284‐28, AI042953, AI45764

Increased TLR Responses in Dendritic Cells Lacking the Itam‐Containing Adapters Dap12 and FcRγ

The inhibitory effect of DAP12 on macrophages has been revealed by examining myeloid cells from DAP12‐deficient mice. In this report, we demonstrate that both DAP12 and the FcεRγ ‐chain (FcRγ) are required for negative regulation of TLR responses in bone marrow‐derived dendritic cells (DCs). Loss of both DAP12 and FcRγ enhanced the pro‐inflammatory cytokine production and maturation of DCs after TLR stimulation, resulting in a greater percentage of DCs that produced IL‐12 p40, TNF, and IL‐6, and expressed high levels of MHC class II, CD80, and CD86. Whereas DCs lacking only DAP12 showed some increased TLR responses, those lacking only FcRγ had a greater enhancement of maturation and cytokine production, though to a lesser extent than DCs lacking both DAP12 and FcRγ Additionally, antigen‐specific T cell activation was enhanced by DAP12−/−FcRγ −/− DCs relative to wild‐type (WT) DCs after maturation. Similar to DAP12−/−FcRγ −/− DCs, Syk‐deficient DCs also had increased inflammatory cytokine production, maturation, and antigen presentation. These results confirm the inhibitory effect of ITAM signaling in myeloid cells and show that DCs and macrophages differ in their dependence on the ITAM‐containing adapters DAP12 and FcRγ for negative regulation of TLR signaling.