Enhancement of TWIK-related acid-sensitive potassium channel 3 (TASK3) two-pore domain potassium channel activity by tumor necrosis factor α.

TASK3 two-pore domain potassium (K2P) channels are responsible for native leak K channels in many cell types which regulate cell resting membrane potential and excitability. In addition, TASK3 channels contribute to the regulation of cellular potassium homeostasis. Because TASK3 channels are important for cell viability, having putative roles in both neuronal apoptosis and oncogenesis, we sought to determine their behavior under inflammatory conditions by investigating the effect of TNFα on TASK3 channel current. TASK3 channels were expressed in tsA-201 cells, and the current through them was measured using whole cell voltage clamp recordings. We show that THP-1 human myeloid leukemia monocytes, co-cultured with hTASK3-transfected tsA-201 cells, can be activated by the specific Toll-like receptor 7/8 activator, R848, to release TNFα that subsequently enhances hTASK3 current. Both hTASK3 and mTASK3 channel activity is increased by incubation with recombinant TNFα (10 ng/ml for 2-15 h), but other K2P channels (hTASK1, hTASK2, hTREK1, and hTRESK) are unaffected. This enhancement by TNFα is not due to alterations in levels of channel expression at the membrane but rather to an alteration in channel gating. The enhancement by TNFα can be blocked by extracellular acidification but persists for mutated TASK3 (H98A) channels that are no longer acid-sensitive even in an acidic extracellular environment. TNFα action on TASK3 channels is mediated through the intracellular C terminus of the channel. Furthermore, it occurs through the ASK1 pathway and is JNK- and p38-dependent. In combination, TNFα activation and TASK3 channel activity can promote cellular apoptosis.

The HIF-1 transcription complex is essential for translational control of myeloid hematopoietic cell function by maintaining mTOR phosphorylation.

Mammalian myeloid cells are crucial effectors of host innate immune defense. Normal and pathological responses of these cells require adaptation to signaling stress through the hypoxia-inducible factor 1 (HIF-1) transcription complex. Adapted cells activate the mammalian target of rapamycin (mTOR), via S2448 phosphorylation, which induces de novo translation of vital signaling proteins. However, the molecular mechanisms underlying this signaling dogma remain unclear. Here, we demonstrate for the first time that inactivation of HIF-1, by silencing its inducible alpha subunit, significantly decreases mTOR S2448 phosphorylation caused by ligand-dependent activation of human myeloid leukemia cells. This shows that HIF-1 is essential for the activation of mTOR and serves at a crucial juncture of myeloid cell function in both in vitro and in vivo systems.

Gold nanoparticles downregulate interleukin-1ß-induced pro-inflammatory responses.

Interleukin 1 beta (IL-1ß)-dependent inflammatory disorders, such as rheumatoid arthritis and psoriasis, pose a serious medical burden worldwide, where patients face a lifetime of illness and treatment. Organogold compounds have been used since the 1930s to treat rheumatic and other IL-1ß-dependent diseases and, though their mechanisms of action are still unclear, there is evidence that gold interferes with the transmission of inflammatory signalling. Here we show for the first time that citrate-stabilized gold nanoparticles, in a size dependent manner, specifically downregulate cellular responses induced by IL-1ß both in vitro and in vivo. Our results indicate that the anti-inflammatory activity of gold nanoparticles is associated with an extracellular interaction with IL-1ß, thus opening potentially novel options for further therapeutic applications.

T lymphocytes induce human cancer cells derived from solid malignant tumors to secrete galectin-9 which facilitates immunosuppression in cooperation with other immune checkpoint proteins.

BACKGROUND: Galectin-9 is a member of the family of lectin proteins and crucially regulates human immune responses, particularly because of its ability to suppress the anticancer activities of T lymphocytes and natural killer cells. Recent evidence demonstrated that galectin-9 is highly expressed in a wide range of human malignancies including the most aggressive tumors, such as high-grade glioblastomas and pancreatic ductal adenocarcinomas, as well as common malignancies such as breast, lung and colorectal cancers. However, solid tumor cells at rest are known to secrete either very low amounts of galectin-9 or, in most of the cases, do not secrete it at all. Our aims were to elucidate whether T cells can induce galectin-9 secretion in human cancer cells derived from solid malignant tumors and whether this soluble form displays higher systemic immunosuppressive activity compared with the cell surface-based protein. METHODS: A wide range of human cancer cell lines derived from solid tumours, keratinocytes and primary embryonic cells were employed, together with helper and cytotoxic T cell lines and human as well as mouse primary T cells. Western blot analysis, ELISA, quantitative reverse transcriptase-PCR, on-cell Western and other measurement techniques were used to conduct the study. Results were validated using in vivo mouse model. RESULTS: We discovered that T lymphocytes induce galectin-9 secretion in various types of human cancer cells derived from solid malignant tumors. This was demonstrated to occur via two differential mechanisms: first by translocation of galectin-9 onto the cell surface followed by its proteolytic shedding and second due to autophagy followed by lysosomal secretion. For both mechanisms a protein carrier/trafficker was required, since galectin-9 lacks a secretion sequence. Secreted galectin-9 pre-opsonised T cells and, following interaction with other immune checkpoint proteins, their activity was completely attenuated. As an example, we studied the cooperation of galectin-9 and V-domain Ig-containing suppressor of T cell activation (VISTA) proteins in human cancer cells. CONCLUSION: Our results underline a crucial role of galectin-9 in anticancer immune evasion. As such, galectin-9 and regulatory pathways controlling its production should be considered as key targets for immunotherapy in a large number of cancers.

L-Kynurenine participates in cancer immune evasion by downregulating hypoxic signaling in T lymphocytes.

Malignant tumors often escape anticancer immune surveillance by suppressing the cytotoxic functions of T lymphocytes. While many of these immune evasion networks include checkpoint proteins, small molecular weight compounds, such as the amino acid L-kynurenine (LKU), could also substantially contribute to the suppression of anti-cancer immunity. However, the biochemical mechanisms underlying the suppressive effects of LKU on T-cells remain unclear. Here, we report for the first time that LKU suppresses T cell function as an aryl hydrocarbon receptor (AhR) ligand. The presence of LKU in T cells is associated with AhR activation, which results in competition between AhR and hypoxia-inducible factor 1 alpha (HIF-1α) for the AhR nuclear translocator, ARNT, leading to T cell exhaustion. The expression of indoleamine 2,3-dioxygenase 1 (IDO1, the enzyme that leads to LKU generation) is induced by the TGF-ß-Smad-3 pathway. We also show that IDO-negative cancers utilize an alternative route for LKU production via the endogenous inflammatory mediator, the high mobility group box 1 (HMGB-1)-interferon-gamma (IFN-γ) axis. In addition, other IDO-negative tumors (like T-cell lymphomas) trigger IDO1 activation in eosinophils present in the tumor microenvironment (TME). These mechanisms suppress cytotoxic T cell function, and thus support the tumor immune evasion machinery.

Involvement of xanthine oxidase and hypoxia-inducible factor 1 in Toll-like receptor 7/8-mediated activation of caspase 1 and interleukin-1ß.

Inflammatory reactions to ssRNA viruses are induced by the endosomal Toll-like receptors (TLRs) 7 and 8. TLR7/8-mediated inflammatory reaction results in activation of the Nalp3 inflammasome via an unknown mechanism. Here we report for the first time that TLR7/8 mediate activation of xanthine oxidase (XOD) in an HIF-1α-dependent manner. XOD produces uric acid and reactive oxygen species, which could activate Nalp3 and therefore induce activation of caspase 1, known to convert inactive pro-IL-1ß into active IL-1ß. Specific inhibition of the XOD activity attenuates TLR7/8-mediated activation of caspase 1 and IL-1ß release. These results were obtained using human THP-1 myeloid macrophages. The findings were verified by conducting in vivo experiments on mice.

IgE-dependent human basophil responses are inversely associated with the sarcoplasmic reticulum Ca2+-ATPase (SERCA).

Basophils crucially contribute to allergies and other Th2-driven diseases by rapidly releasing inflammatory and immunomodulatory mediators following high-affinity IgE-receptor crosslinking. Although these basophil-mediated responses depend on sensitization with antigen-specific IgE, this does not necessarily predict clinical symptom severity. It is thought that the balance of early stimulatory (e.g. SYK) and inhibitory (e.g. SHIP-1) intracellular signals are associated with basophil responsiveness, which is also critically dependent on calcium mobilization. Previous studies suggest that the sarcoplasmic reticulum Ca2+-ATPase (SERCA2), which regulates cytosolic calcium levels, may be inversely associated with airway smooth muscle reactivity in asthma. Since basophils are implicated in asthma severity, our aims were to address whether SERCA2 is implicated in human basophil responses, especially following IgE-mediated activation. Human basophils were obtained from buffy coats, following research ethics approval, and further purified by immunomagnetic cell sorting. Expressions of SERCA2, and other isoforms, were determined by Western blotting in parallel to measuring IgE-dependent histamine releases from the same donors. The effects of a SERCA-activator and inhibitor were also assessed on their abilities to modulate basophil histamine release. We observed an inverse correlation between basophil responsiveness to IgE-dependent stimulation and SERCA2 expression. Thapsigargin, a highly-specific SERCA inhibitor, stimulated basophil histamine release and potentiated IgE-dependent secretion of the amine. Conversely, disulfiram, a SERCA activator, inhibited IgE-dependent basophil activation. The results obtained from this exploratory study indicate that SERCA2 may be an additional regulator of basophil reactivity alongside early excitatory or inhibitory signal transduction pathways.

Expression of the Immune Checkpoint Protein VISTA Is Differentially Regulated by the TGF-ß1 - Smad3 Signaling Pathway in Rapidly Proliferating Human Cells and T Lymphocytes.

Immune checkpoint proteins play crucial roles in human embryonic development but are also used by cancer cells to escape immune surveillance. These proteins and biochemical pathways associated with them form a complex machinery capable of blocking the ability of cytotoxic immune lymphoid cells to attack cancer cells and, ultimately, to fully suppress anti-tumor immunity. One of the more recently discovered immune checkpoint proteins is V-domain Ig-containing suppressor of T cell activation (VISTA), which plays a crucial role in anti-cancer immune evasion pathways. The biochemical mechanisms underlying regulation of VISTA expression remain unknown. Here, we report for the first time that VISTA expression is controlled by the transforming growth factor beta type 1 (TGF-ß)-Smad3 signaling pathway. However, in T lymphocytes, we found that VISTA expression was differentially regulated by TGF-ß depending on their immune profile. Taken together, our results demonstrate the differential biochemical control of VISTA expression in human T cells and various types of rapidly proliferating cells, including cancer cells, fetal cells and keratinocytes.

Macrophage Differentiation and Polarization Regulate the Release of the Immune Checkpoint Protein V-Domain Ig Suppressor of T Cell Activation.

Recently, the V-domain immunoglobulin suppressor of T-cell activation (VISTA) was identified as a negative immune checkpoint regulator (NCR) that is mainly expressed in hematopoietic cells. Preclinical studies have shown that VISTA blockade results in impeded tumor growth and improved survival. Nevertheless, little is known about the physiological role of VISTA expression in macrophages. This study focused on the differential expression of VISTA in human monocytes and macrophages in order to elucidate a putative role of VISTA regulation upon macrophage polarization and activation. We observed that human peripheral monocytes constitutively release soluble VISTA, which was regulated via matrix metalloproteinases. However, monocyte stimulation with cytokines that induce macrophage differentiation, such as granulocyte-macrophage colony-stimulating (GM-CSF) and macrophage colony-stimulating factor (M-CSF), substantially reduced soluble VISTA release. VISTA release was further affected by various pro- and anti-inflammatory stimuli that led to macrophage polarization, where activated M1 macrophages generally released more VISTA than M2 macrophages. Additionally, we observed that stimulation of activated macrophages with the toll-like receptor 4 ligand lipopolysaccharide (LPS) led to a further decrease of soluble VISTA release. Moreover, we found that soluble VISTA impairs T cell cytotoxic activity but did not induce their programmed death. Our results suggest that VISTA is constantly produced and released in the peripheral blood where it may contribute to peripheral tolerance.

The involvement of sphingosine kinase 1 in LPS-induced Toll-like receptor 4-mediated accumulation of HIF-1α protein, activation of ASK1 and production of the pro-inflammatory cytokine IL-6.

Toll-like receptors (TLRs) lie in the core of resistance to infectious diseases allowing host immune cells to specifically detect pathogens by recognising their specific molecular patterns. Cell membrane-associated TLR4 (recognises lipopolysaccharide (LPS) of Gram-negative bacteria) and endosomal TLR7/8 (recognise viral single-stranded RNA) are known to activate hypoxia inducible factor-1α (HIF-1α) protein (necessary for cellular adaptation to the inflammatory stress) via redox-dependent mechanism. TLR4 triggers the cross talk between HIF-1α and apoptosis signal-regulating kinase 1 (ASK1), whereas TLR7/8 activates HIF-1α in the ASK1-independent manner. Here, we report that in THP-1 and RAW264.7 macrophages, ligand-induced activation of the TLR4 but not TLR7/8 induces activation and transcriptional upregulation of sphingosine kinase 1 (SphK1) in extracellular signal-regulating kinase and phospholipase C-1γ/PI3 kinase-dependent manner. TLR4-mediated SphK1 activation was found to be critical for the redox-dependent activation of HIF-1α and ASK1, as well as for the prevention of LPS-induced activation of caspase 3 and the expression of pro-inflammatory cytokine interleukin-6.

Functional role of galectin-9 in directing human innate immune reactions to Gram-negative bacteria and T cell apoptosis.

Galectin-9 is a member of the galectin family of proteins, which were first identified to specifically bind to carbohydrates containing ß-galactosides. Galectin-9 is conserved through evolution and recent evidence demonstrated its involvement in innate immune reactions to bacterial infections as well as the suppression of cytotoxic immune responses of T and natural killer cells. However, the molecular mechanisms underlying such differential immunological functions of galectin-9 remain largely unknown. In this work we confirmed that soluble galectin-9 derived from macrophages binds to Gram-negative bacteria by interacting with lipopolysaccharide (LPS), which forms their cell wall. This opsonisation effect most likely interferes with the mobility of bacteria leading to their phagocytosis by innate immune cells. Galectin-9-dependent opsonisation also promotes the innate immune reactions of macrophages to these bacteria and significantly enhances the production of pro-inflammatory cytokines - interleukin (IL) 6, IL-1ß and tumour necrosis factor alpha (TNF-α). In contrast, galectin-9 did not bind peptidoglycan (PGN), which forms the cell wall of Gram-positive bacteria. Moreover, galectin-9 associated with cellular surfaces (studied in primary human embryonic cells) was not involved in the interaction with bacteria or bacterial colonisation. However, galectin-9 expressed on the surface of primary human embryonic cells, as well as soluble forms of galectin-9, were able to target T lymphocytes and caused apoptosis in T cells expressing granzyme B. Furthermore, "opsonisation" of T cells by galectin-9 led to the translocation of phosphatidylserine onto the cell surface and subsequent phagocytosis by macrophages through Tim-3, the receptor, which recognises both galectin-9 and phosphatidylserine as ligands.

High Mobility Group Box 1 (HMGB1) Induces Toll-Like Receptor 4-Mediated Production of the Immunosuppressive Protein Galectin-9 in Human Cancer Cells.

High mobility group box 1 (HMGB1) is a non-histone protein which is predominantly localised in the cell nucleus. However, stressed, dying, injured or dead cells can release this protein into the extracellular matrix passively. In addition, HMGB1 release was observed in cancer and immune cells where this process can be triggered by various endogenous as well as exogenous stimuli. Importantly, released HMGB1 acts as a so-called "danger signal" and could impact on the ability of cancer cells to escape host immune surveillance. However, the molecular mechanisms underlying the functional role of HMGB1 in determining the capability of human cancer cells to evade immune attack remain unclear. Here we report that the involvement of HMGB1 in anti-cancer immune evasion is determined by Toll-like receptor (TLR) 4, which recognises HMGB1 as a ligand. We found that HGMB1 induces TLR4-mediated production of transforming growth factor beta type 1 (TGF-ß), displaying autocrine/paracrine activities. TGF-ß induces production of the immunosuppressive protein galectin-9 in cancer cells. In TLR4-positive cancer cells, HMGB1 triggers the formation of an autocrine loop which induces galectin-9 expression. In malignant cells lacking TLR4, the same effect could be triggered by HMGB1 indirectly through TLR4-expressing myeloid cells present in the tumour microenvironment (e. g. tumour-associated macrophages).

Involvement of hypoxia-inducible factor-1 HiF(1alpha) in IgE-mediated primary human basophil responses.

Basophils play a pivotal role in regulating chronic allergic inflammation as well as angiogenesis. Here, we show for the first time that IgE-mediated activation of primary human basophils results in protein accumulation of the alpha-subunit of hypoxia-inducible factor 1alpha (HIF-1alpha), which is differentially regulated compared with signals controlling histamine release. HIF-1 facilitates cellular adaptation to hypoxic conditions such as inflammation and tumour growth by controlling glycolysis, angiogenesis and cell adhesion. ERK and p38 MAPK, but not reactive oxygen species (ROS), ASK1 or PI 3-kinase, were critical for IgE-mediated accumulation of HIF-1alpha, although the latter crucially affected degranulation. Abrogating HIF-1alpha expression in basophils using siRNA demonstrated that this protein is essential for vascular endothelial growth factor (VEGF) mRNA expression and, consequently, release of VEGF protein. In addition, HIF-1alpha protein alters IgE-induced ATP depletion in basophils, thus also supporting the production of the pro-allergic cytokine IL-4.

The role of redox-dependent mechanisms in the downregulation of ligand-induced Toll-like receptors 7, 8 and 4-mediated HIF-1 alpha prolyl hydroxylation.

Toll-like receptors (TLRs) are key components of the innate immune system that allow immune cells to specifically detect pathogens by recognizing their specific molecular patterns. Hypoxia-inducible factor-1 alpha (HIF-1 alpha) is known to have a critical role in TLR downstream signalling by promoting energy metabolism, expression of proinflammatory cytokines and proangiogenic factors. However, the molecular mechanisms leading to the accumulation of HIF-1 alpha are not fully understood. In this study, we report that R848 (specific ligand)-induced activation of endosomal TLRs 7 and 8 (which recognize viral single-stranded RNA) and lipopolysaccharide (LPS)-induced activation of TLR4 (which specifically recognizes LPS as a ligand) leads to downregulation of degradative HIF-1 alpha prolyl hydroxylation. In the case of TLR7/8, this downregulation is achieved through redox- and reactive nitrogen species (RNS)-dependent mechanisms. S-nitrosation of HIF-1 alpha protein was also observed. In the case of LPS-induced TLR4 activation, only a redox-dependent mechanism is involved. RNS and p38 MAP kinase (known to contribute to LPS-induced TLR4-dependent accumulation of HIF-1 alpha protein) do not affect HIF-1 alpha prolyl hydroxylation. In both cases, downregulation of HIF-1 alpha prolyl hydroxylation correlates with a decrease in intracellular iron (II).

Gene Silencing Approaches in Mast Cells and Primary Human Basophils.

The ability to silence gene expression is an invaluable tool for elucidating the importance of intracellular signaling proteins which contribute to the effector functions of mast cells and basophils. However, primary mast cells and their terminally differentiated blood counterpart, basophils, pose a difficult challenge for gene silencing approaches given not only their state of maturation and difficulty to transfect but also because their functions are readily altered by cell handling conditions. Here, we describe a method using lipofection which has been successfully employed to silence gene expression using siRNA in human LAD2 mast cells as well as primary human basophils.

Ligand-Receptor Interactions of Galectin-9 and VISTA Suppress Human T Lymphocyte Cytotoxic Activity.

Acute myeloid leukemia (AML), a blood/bone marrow cancer, is a severe and often fatal malignancy. AML cells are capable of impairing the anti-cancer activities of cytotoxic lymphoid cells. This includes the inactivation of natural killer (NK) cells and killing of T lymphocytes. Here we report for the first time that V-domain Ig-containing suppressor of T cell activation (VISTA), a protein expressed by T cells, recognizes galectin-9 secreted by AML cells as a ligand. Importantly, we found that soluble VISTA released by AML cells enhances the effect of galectin-9, most likely by forming multiprotein complexes on the surface of T cells and possibly creating a molecular barrier. These events cause changes in the plasma membrane potential of T cells leading to activation of granzyme B inside cytotoxic T cells, resulting in apoptosis.

Transforming growth factor beta type 1 (TGF-ß) and hypoxia-inducible factor 1 (HIF-1) transcription complex as master regulators of the immunosuppressive protein galectin-9 expression in human cancer and embryonic cells.

Galectin-9 is one of the key proteins employed by a variety of human malignancies to suppress anti-cancer activities of cytotoxic lymphoid cells and thus escape immune surveillance. Human cancer cells in most cases express higher levels of galectin-9 compared to non-transformed cells. However, the biochemical mechanisms underlying this phenomenon remain unclear. Here we report for the first time that in human cancer as well as embryonic cells, the transcription factors hypoxia-inducible factor 1 (HIF-1) and activator protein 1 (AP-1) are involved in upregulation of transforming growth factor beta 1 (TGF-ß1) expression, leading to activation of the transcription factor Smad3 through autocrine action. This process triggers upregulation of galectin-9 expression in both malignant (mainly in breast and colorectal cancer as well as acute myeloid leukaemia (AML)) and embryonic cells. The effect, however, was not observed in mature non-transformed human cells. TGF-ß1-activated Smad3 therefore displays differential behaviour in human cancer and embryonic vs non-malignant cells. This study uncovered a self-supporting biochemical mechanism underlying high levels of galectin-9 expression operated by the human cancer and embryonic cells employed in our investigations. Our results suggest the possibility of using the TGF-ß1 signalling pathway as a potential highly efficient target for cancer immunotherapy.

HIF-1alpha protein is an essential factor for protection of myeloid cells against LPS-induced depletion of ATP and apoptosis that supports Toll-like receptor 4-mediated production of IL-6.

Sepsis is the leading cause of death in intensive care units, which reflects detrimental host response to infection where lipopolysaccharide (LPS) shared by Gram-negative bacteria acts as a potent activator of immune cells via Toll-like receptor 4 (TLR4). Recently it was found that TLR4 downstream signalling leads to the accumulation of hypoxia-inducible factor 1 alpha (HIF-1alpha), which is important for TLR4-dependent expression of pro-inflammatory cytokines, however, basic biochemical mechanisms of involvement of this protein in TLR4 downstream signalling remains unclear. Here we found that knockdown of the expression of HIF-1alpha protein by siRNA led to the depletion of ATP, which corresponded to the constant increase in the activity of apoptosis signal-regulating kinase 1 (ASK1) and therefore apoptosis as estimated based on the increase in the activity of caspase 3. On the other hand, LPS-dependent production of IL-6 was attenuated. Treatment of HIF-1alpha knockdown cells with extracellular ATP in combination with LPS preserved the IL-6 expression but not the activity of ASK1 on the level observed in LPS-stimulated control cells. We therefore suggested that HIF-1alpha protein supports LPS-dependent expression of IL-6 by preventing depletion of ATP. On the other hand HIF-1alpha protein is selectively required for down-regulation of ASK1 activated during LPS-induced TLR4 downstream signalling.

Mitochondrial Defunctionalization Supresses Tim-3-Galectin-9 Secretory Pathway in Human Colorectal Cancer Cells and Thus Can Possibly Affect Tumor Immune Escape.

The Tim-3-galectin-9 secretory pathway is known to protect various types of cancer cells against host immune surveillance. We found that pharmacologically induced mitochondrial dysfunction leads to a reduced galectin-9 expression/exocytosis in human colorectal cancer cells and re-distribution of this protein (the effect described for various cellular proteins) into mitochondria.

Targeting of Basophil and Mast Cell Pro-Allergic Reactivity Using Functionalised Gold Nanoparticles.

Calcineurin inhibitors potentially prevent pro-allergic mediator release from basophils and mast cells but are rarely used systemically due to ubiquitous expressions of target signaling proteins. However, specific targeting of allergic effector cells with these inhibitors could circumvent unwanted side effects. We recently demonstrated the biocompatibility of gold nanoparticles (AuNPs) as a platform for non-toxic delivery of signaling inhibitors due to unique physicochemical properties of these nanomaterials. Since AuNPs can be conjugated with both anti-allergic drugs and antibodies or other proteins that specifically recognize basophils and mast cells, our aims were to assess specific targeting of allergic effector cell function using AuNPs conjugated with the calcineurin inhibitor ascomycin. Purified human basophils and LAD2 human mast cells were used for investigations with AuNPs conjugated either to CD203c antibodies or containing stem cell factor (SCF), respectively, which were amine-coupled to acidic groups of reduced glutathione (GSH). GSH was also used as a spacer for immobilization of ascomycin on the gold surface. AuNPs conjugated with anti-CD203c and ascomycin strikingly blocked IgE-dependent degranulation of both purified basophils and those present in mixed leukocyte preparations, suggesting specific targeting of these cells. In contrast, LAD2 mast cell responses were not inhibited using anti-CD203c-containing nanoconjugates but were when the conjugates contained SCF. Successful targeting of allergic effector cells using gold nanoconjugates indicates that this technology may have therapeutic potential for the treatment of allergies by specifically delivering highly effective signaling inhibitors with reduced side effects.