T cell activation induced by novel gain-of-function mutants of Syk and ZAP-70

The Syk family tyrosine kinases play a crucial role in antigen receptor-mediated signal transduction, but their regulation and cellular targets remain incompletely defined. Following receptor engagement, phosphorylation of tyrosine residues within ZAP-70 and Syk is thought to control both kinase activity and recruitment of modulatory factors. We report here the characterization of novel mutants of ZAP-70 and Syk, in which conserved C-terminal tyrosine residues have been replaced by phenylalanines (ZAP YF-C, Syk YF-C). Both mutant kinases display a prominent gain-of-function phenotype in Jurkat T cells, as demonstrated by lymphokine promoter activation, tyrosine phosphorylation of potential targets in vivo, and elevated intracellular calcium mobilization. While the presence of p56-Lck was required for ZAP YF-C-induced signaling, Syk YF-C showed enhanced functional activity in Lck-deficient JCaM1 Jurkat cells. Our results implicate the C terminus of Syk family kinases as an important regulatory region modulating T cell activation.     

Differential requirements for ZAP-70 in TCR signaling and T cell development

The Syk/ZAP-70 family of protein tyrosine kinases is indispensable for normal lymphoid development. Syk is necessary for the development of B cells and epithelial gammadelta T cells, whereas ZAP-70 is essential for the normal development of T cells and TCR signaling. In this study, we show that although development of the alphabeta lineage was arrested in the thymus, CD3-positive T cells, primarily of the gammadelta lineage, were present in the lymph nodes of mice lacking ZAP-70. Moreover, in the absence of ZAP-70, dendritic epidermal T cells were fewer in number and of abnormal morphology, and intestinal intraepithelial lymphocytes, normally containing a large proportion of gammadelta T cells, were markedly reduced. These data suggest that gammadelta T cells show a variable dependence upon ZAP-70 for their development. Biochemical analyses of thymocytes revealed a lack of basal zeta-chain tyrosine phosphorylation. However, several other substrates were inducibly tyrosine phosphorylated following TCR stimulation. Thus, TCR-mediated signaling in ZAP-70-deficient thymocytes is only partially impaired. These studies suggest that Syk compensates only partially for the loss of ZAP-70, and that there is an absolute requirement of ZAP-70 for alphabeta T cells and epithelial gammadelta T cells, but not for some gammadelta T cells in peripheral lymphoid tissues.     

Human immunodeficiency virus infection abolishes CD4-dependent activation of ZAP-70 by inhibition of p56lck

The effect of early human immunodeficiency virus-1 infection in vitro on proximal signal transduction events in primary peripheral blood lymphocytes was investigated with respect to CD4-mediated costimulation of CD3/T cell-receptor signalling. Tyrosine phosphorylation profiles induced by CD4 and CD3 + CD4 ligation were profoundly abrogated in virally infected cells, although CD4 receptor expression remained intact during early infection. Furthermore, the association of the tyrosine kinase p56lck with the CD4 receptor was reduced in virally infected cells. The downmodulation of CD4-mediated CD3 signalling coincided with the subsequent inhibition of the activity and tyrosine phosphorylation of the downstream kinase ZAP-70 in virally infected cells. The observed virally mediated cosignalling defects during early infection may account for the inhibition of distal signal events and thus contribute to HIV pathogenesis, such as reduced immune response to antigenic exposure, anergy, and apoptosis.     

IL-10 inhibits macrophage activation and proliferation by distinct signaling mechanisms: evidence for Stat3-dependent and -independent pathways

Interleukin-10 (IL-10) limits inflammatory responses by inhibiting macrophage activation. In macrophages, IL-10 activates Stat1 and Stat3. We characterized IL-10 responses of the J774 mouse macrophage cell line, and of J774 cells expressing wild-type hIL-10R, mutant hIL-10R lacking two membrane-distal tyrosines involved in recruitment of Stat3 (hIL-10R-TyrFF), a truncated Stat3 (DeltaStat3) which acts as a dominant negative, or an inducibly active Stat3-gyraseB chimera (Stat3-GyrB). A neutralizing anti-mIL-10R monoclonal antibody was generated to block the function of endogenous mIL-10R. IL-10 inhibited proliferation of J774 cells and of normal bone marrow-derived macrophages, but not J774 cells expressing hIL-10RTyrFF. Dimerization of Stat3-GyrB by coumermycin mimicked the effect of IL-10, and expression of DeltaStat3 blocked the anti-proliferative activity of IL-10. For macrophage de-activation responses, hIL10R-TyrFF could not mediate inhibition of lipopolysaccharide-induced TNFalpha, IL-1beta or CD86 expression, while DeltaStat3 did not interfere detectably with these IL-10 responses. Thus signals mediating both anti-proliferative and macrophage de-activation responses to IL-10 require the two membrane-distal tyrosines of IL-10R, but Stat3 appears to function only in the anti-proliferative response.     

A dual participation of ZAP-70 and scr protein tyrosine kinases is required for TCR-induced tyrosine phosphorylation of Sam68 in Jurkat T cells

Sam68 has been initially described as a substrate of src kinases during mitosis in fibroblasts. Recent evidence suggests that in T lymphocytes Sam68 may act as an adaptor protein and participate in the early biochemical cascade triggered after CD3 stimulation. A direct interaction between Sam68 and the two src kinases involved in T cell activation, p59(fyn) and p56(lck), as well as a partnership of Sam68 with various key downstream signaling molecules, like phospholipase Cgamma-1 and Grb2, has been shown. In this study we analyze the contribution of p56(lck), as well as the role of ZAP-70, the second class of protein tyrosine kinase involved in T cell activation, in Sam68 tyrosine phosphorylation in the human Jurkat T cell line. Using the src inhibitor PP1 [4-amino-5-(4-methylphenyl)7-(t-butyl) pyrazolo [3,4-d] pyrymidine] and cell variants with defective expression of p56(lck) or expressing a dominant negative form of ZAP-70, we demonstrate that, while both p56(lck) and ZAP-70 are dispensable for the low constitutive phosphorylation of Sam68 observed in Jurkat cells, a cooperation between the two kinases is required to increase its rapid phosphorylation observed in vivo after CD3 stimulation. We also show that recombinant forms of both p56(lck) and ZAP-70 phosphorylate Sam68 in vitro. However, using CD2 stimulated cells, we observe that p56(lck) activation by itself does not induce Sam68 tyrosine phosphorylation. We conclude that p59(fyn) and p56(lck) differently participate in regulating the phosphorylation state of Sam68 in T cells and that ZAP-70 may contribute to Sam68 tyrosine phosphorylation and to the specific recruitment of this molecule after CD3 stimulation.     

CD28-independent, TRAF2-dependent costimulation of resting T cells by 4-1BB ligand

4-1BB ligand (4-1BBL) is a member of the tumor necrosis factor (TNF) family expressed on activated antigen-presenting cells. Its receptor, 4-1BB, is a member of the TNF receptor family expressed on activated CD4 and CD8 T cells. We have produced a soluble form of 4-1BBL using the baculovirus expression system. When coimmobilized on plastic with anti-CD3, soluble 4-1BBL induces interleukin (IL)-2 production by resting CD28+ or CD28- T cells, indicating that 4-1BBL can function independently of other cell surface molecules, including CD28, in costimulation of resting T cell activation. At low concentrations of anti-CD3, 4-1BBL is inferior to anti-CD28 in T cell activation. However, when 4-1BB ligand is provided together with strong TCR signals, then 4-1BBL and anti-CD28 are equally potent in stimulation of IL-2 production by resting T cells. We find that TNF receptor-associated factor (TRAF)1 or TRAF2 associate with a glutathione S-transferase-4-1BB cytoplasmic domain fusion protein in vitro. In T cells, we find that association of TRAF1 and TRAF2 with 4-1BB requires 4-1BB cross-linking. In support of a functional role for TRAF2 in 4-1BB signaling, we find that resting T cells isolated from TRAF2-deficient mice or from mice expressing a dominant negative form of TRAF2 fail to augment IL-2 production in response to soluble 4-1BBL. Thus 4-1BB, via the TRAF2 molecule, can provide CD28-independent costimulatory signals to resting T cells.     

Ca2+-dependent K+ currents induced by muscarinic receptor activation in guinea pig adrenal chromaffin cells

The neuronal effects of the metabotropic glutamate receptor agonist (1S,3R)-aminocyclopentane-1,3-dicarboxylic acid have been studied in cultured rat cerebellar granule cells, and compared with those of the endogenous excitotoxin glutamate, and the dietary excitotoxin beta-N-methylamino-L-alanine. Glutamate, beta-N-methylamino-L-alanine, and (1S,3R)-aminocyclopentane-1,3-dicarboxylic acid all caused concentration-dependent cerebellar granule cell death over a 24-h exposure period. The metabotropic antagonist (RS)-alpha-methyl-4-carboxyphenylglycine reduced glutamate-, beta-N-methylamino-L-alanine-, and (1S,3R)-aminocyclopentane-1,3-dicarboxylic acid-induced death by 50, 37, and 90%, respectively. (1S,3R)-Aminocyclopentane-1,3-dicarboxylic acid-induced death was unaffected by the group I antagonist (RS)-1-aminoindan-1,5-dicarboxylic acid, increased by the group II antagonist ethylglutamic acid, and markedly decreased by the group III antagonist (RS)-alpha-methylserine-O-phosphate. Neither (1S,3R)-aminocyclopentane-1,3-dicarboxylic acid nor the group I agonist (RS)-3,5-dihydroxyphenylglycine caused an increase in intracellular free calcium levels. The group III agonist L-(+)-2-amino-4-phosphonobutyric acid also induced concentration-dependent cerebellar granule cell death, and so it was suggested that the group III metabotropic glutamate receptors were responsible for (1S,3R)-aminocyclopentane-1,3-dicarboxylic acid-induced death. Blocking these receptors with (RS)-alpha-methylserine-O-phosphate also prevented a proportion of glutamate- and beta-N-methylamino-L-alanine-induced death.     

Calmodulin regulation of Ca2+ entry in Jurkat T cells

BACKGROUND: The authors have previously demonstrated abnormalities in glucose and insulin metabolism in nondiabetic black American (BA) adults versus white American (WA) adults. Whether similar glucoregulatory alterations extend to BA adolescents remain unknown. In addition, obesity, a known risk factor for insulin resistance and hyperinsulinemia, occurs in a greater proportion of BA adults and children when compared to WA. The objective of the present study was to examine the differential effects of obesity on glucose homeostasis in BA and WA adolescents. METHODS: We examined glucose homeostasis in BA and WA adolescents using oral glucose tolerance test (OGTT), intravenous glucose tolerance test (IVGTT), and [6,6-2H2]-glucose infusion. The study consisted of four age-, sex-, and pubertal stage-matched groups: 15 lean BA, 29 lean WA, 7 obese BA, and 9 obese WA. RESULTS: Both obese groups had significantly increased insulin and C-peptide area under the curve (AUC) during OGTT and IVGTT when compared to their same-race lean counterparts. During OGTT, obese BA demonstrated greater insulin and C-peptide when compared to obese WA. During IVGTT, first- and second-phase insulin were significantly greater in obese BA versus obese WA. CONCLUSION: In summary, BA adolescents demonstrated insulin resistance which is markedly exaggerated in the face of obesity when compared to WA adolescents, implying a differential impact for obesity on glucose homeostasis that is unique to the obese BA adolescent group. In conclusion, there is a need for early aggressive weight management in obese BA adolescents.     

Antigen-dependent and -independent Ca2+ responses triggered in T cells by dendritic cells compared with B cells

Dendritic cells (DCs) are much more potent antigen (Ag)-presenting cells than resting B cells for the activation of naive T cells. The mechanisms underlying this difference have been analyzed under conditions where ex vivo DCs or B cells presented known numbers of specific Ag-major histocompatibility complex (MHC) complexes to naive CD4(+) T cells from T cell antigen receptor (TCR) transgenic mice. Several hundred Ag-MHC complexes presented by B cells were necessary to elicit the formation of a few T-B conjugates with small contact zones, and the resulting individual T cell Ca2+ responses were all-or-none. In contrast, Ag-specific T cell Ca2+ responses can be triggered by DCs bearing an average of 30 Ag-MHC complexes per cell. Formation of T-DC conjugates is Ag-independent, but in the presence of the Ag, the surface of the contact zone increases and so does the amplitude of the T cell Ca2+ responses. These results suggest that Ag is better recognized by T cells on DCs essentially because T-DC adhesion precedes Ag recognition, whereas T-B adhesion requires Ag recognition. Surprisingly, we also recorded small Ca2+ responses in T cells interacting with unpulsed DCs. Using DCs purified from MHC class II knockout mice, we provide evidence that this signal is mostly due to MHC-TCR interactions. Such an Ag-independent, MHC-triggered calcium response could be a survival signal that DCs but not B cells are able to deliver to naive T cells.     

Ca(2+)-dependent and Ca(2+)-independent isozymes of protein kinase C mediate exocytosis in antigen-stimulated rat basophilic RBL-2H3 cells. Reconstitution of secretory responses with Ca2+ and purified isozymes in washed permeabilized cells

Rat basophilic RBL-2H3 cells, which exhibit Ca(2+)-dependent secretion of granules when stimulated with antigen, contained the Ca(2+)-dependent alpha and beta and the Ca(2+)-independent delta, epsilon, and zeta isoforms of protein kinase C. These isoforms associated, to variable extents (i.e. delta the most and zeta the least), with the membrane fraction upon antigen stimulation but without external Ca2+; only the Ca(2+)-independent isoforms did so. Both types of isozymes were probably necessary for optimal responses to antigen as indicated by the following observations. All Ca(2+)-dependent isozymes were degraded in cells treated with 20 nM phorbol 12-myristate 13-acetate for 6 h, whereas the Ca(2+)-independent isozymes were not degraded and were retained when the cells were subsequently permeabilized and washed. Cells so treated still exhibited antigen-induced secretion (25-33% of normal) which was suppressed by selective inhibitors of protein kinase C (Ro31-7549 and calphostin C) thereby indicating a possible contribution of the Ca(2+)-independent isozymes in secretion. Normally, washed permeabilized cells lost all isozymes of protein kinase C and failed to secrete in response to antigen. A full secretory response to antigen could be reconstituted by the subsequent addition of nanomolar concentrations of either beta or delta isozymes of protein kinase C (other isozymes were much less effective) but only in the presence of 1 microM free Ca2+ to indicate distinct roles for Ca2+ and protein kinase C in exocytosis.     

Histamine increases anti-CD3 induced IL-5 production of TH2-type T cells via histamine H2-receptors

Besides its proinflammatory functions histamine released from basophils and mast cells during immediate-type hypersensitivity reactions is known to inhibit several lymphocyte functions like IL-2 and gamma-IFN production. Recently, it has been shown that T helper cells of type 2 phenotype (TH2) represent the T cell fraction which may play a pivotal role in the promotion of the allergic inflammatory eosinophilic late-phase reaction by secretion of cytokines, especially IL-4 and IL-5. We have investigated the effect of histamine on anti-CD3 induced IL-4 and IL-5 production by TH2 cells. Histamine in concentrations between 10(-7) and 10(-5) mol/l concentration-dependently increased anti-CD3 induced IL-5 production up to 120%, whereas IL-4 production was not affected. The activity of histamine in increasing IL-5 production was mimicked by the H2-receptor agonist dimaprit. Histamine induced increase in IL-5 production was inhibited by histamine H2-receptor antagonists, but remained unaffected by H1- or H3-receptor antagonists. Administration of forskolin which directly stimulates the production of cAMP, the second messenger of the H2-receptor, also resulted in an increase in anti-CD3 induced IL-5 production. These results indicate that the histamine-mediated increase in anti-CD3 induced IL-5 production is mediated via H2-receptors. Consequently, histamine released from mast cells and basophils during the early-phase allergic reaction may act as an important stimulatory signal for the initiation of the allergic inflammatory late-phase reaction by increasing local IL-5 production of allergen triggered TH2 cells.     

Involvement of phosphoinositide 3-kinase and Rac in membrane ruffling induced by IL-2 in T cells

IL-2 is known to play a critical role in regulating T lymphocyte proliferation. We show here that IL-2 also provokes an instantaneous and sustained membrane ruffling in cloned human or murine T cells as well as in lectin-activated peripheral blood lymphocytes. In the IL-2-induced lamellipodia, tubulin is depolymerized whereas actin is strongly polymerized, forming caps. IL-2-induced membrane ruffling is protein kinase C (PKC) independent, as judged by the absence of effects of bisindolylmaleimide, an efficient inhibitor of all PKC isoforms. The formation of lamellipodia by IL-2 is blocked by wortmannin and LY294002, two inhibitors of phosphoinositide 3-kinase (PI3-kinase). Moreover, expression in murine T cells of an inactive form of P13-kinase inhibits IL-2-induced membrane ruffling, whereas expression of a constitutively active p110 increases the basal membrane ruffling. Rac is also involved in IL-2-induced membrane ruffling since an inactive form of Rac (N17rac) blocks the IL-2-induced lamellipodia, whereas the constitutive form of Rac (Val12rac) can also lead to membrane ruffling. In the signaling cascade, Rac is downstream of PI3-kinase since constitutive membrane ruffling in Val12rac cells is insensitive to wortmannin. Thus, through a signaling cascade involving PI3-kinase and Rac, IL-2 can induce profound alterations of the T cell cytoskeleton, a phenomenon which might be of importance for T cell physiology.     

Neonatal activation of CD28 signaling overcomes T cell anergy and prevents autoimmune diabetes by an IL-4-dependent mechanism

Optimal T cell responsiveness requires signaling through the T cell receptor (TCR) and CD28 costimulatory receptors. Previously, we showed that T cells from autoimmune nonobese diabetic (NOD) mice display proliferative hyporesponsiveness to TCR stimulation, which may be causal to the development of insulin-dependent diabetes mellitus (IDDM). Here, we demonstrate that anti-CD28 mAb stimulation restores complete NOD T cell proliferative responsiveness by augmentation of IL-4 production. Whereas neonatal treatment of NOD mice with anti-CD28 beginning at 2 wk of age inhibits destructive insulitis and protects against IDDM by enhancement of IL-4 production by islet-infiltrating T cells, administration of anti-CD28 beginning at 5-6 wk of age does not prevent IDDM. Simultaneous anti-IL-4 treatment abrogates the preventative effect of anti-CD28 treatment. Thus, neonatal CD28 costimulation during 2-4 wk of age is required to prevent IDDM, and is mediated by the generation of a Th2 cell-enriched nondestructive environment in the pancreatic islets of treated NOD mice. Our data support the hypothesis that a CD28 signal is requisite for activation of IL-4-producing cells and protection from IDDM.     

Involvement of tyrosine phosphorylation and protein kinase C in the activation of phospholipase D by H2O2 in Swiss 3T3 fibroblasts

We have investigated the mechanisms involved in H2O2-mediated phospholipase D (PLD) activation in Swiss 3T3 fibroblasts. In the presence of vanadate, H2O2 induced tyrosine phosphorylation of PLD as well as the platelet-derived growth (PDGF) factor receptor, protein kinase Calpha (PKCalpha), and a 62-kDa protein in rat brain PLD1 (rPLD1) immune complexes. PDGF also induced tyrosine phosphorylation of PLD, but this was abolished by catalase, indicating that it was mediated by H2O2 generation. Interestingly, PLD was found to be constitutively associated with the PDGF receptor and PKCalpha. Stimulation by H2O2 showed a concentration- and time-dependent tyrosine phosphorylation of the proteins in rPLD1 immunoprecipitates and activation of PLD in the cells. Pretreatment of the cells with the protein-tyrosine kinase inhibitors genistein and herbimycin A resulted in a concentration-dependent inhibition of H2O2-induced tyrosine phosphorylation and PLD activation. Activation of PLD by H2O2 was also inhibited dose-dependently by the PKC inhibitors Ro 31-8220 and calphostin C. Down-regulation of PKC by prolonged treatment with 4beta-phorbol 12-myristate 13-acetate also abolished H2O2-stimulated PLD activity. H2O2 or vanadate alone did not induce tyrosine phosphorylation of proteins in the rPLD1 immune complex or PLD activation. Reduction of intracellular H2O2 levels by pretreatment of the cells with catalase dramatically abrogated tyrosine phosphorylation of proteins in the rPLD1 immune complex and PLD activation, suggesting the potential role of intracellular H2O2 in H2O2-mediated PLD signaling. Taken together, these results suggest that both protein-tyrosine kinase(s) and protein kinase C participate in H2O2-induced PLD activation in Swiss 3T3 cells.     

Induction of late activation events by Igbeta signaling subunit of B-cell receptor in Jurkat T-cell without tyrosine phosphorylation

T-lymphocyte activation consists of multiple intracellular signaling events, eventually leading to cellular proliferation by the control of cytokine gene expression and the acquisition of diverse effector function. To investigate the functional specificity of ITAM (Immunoreceptor Tyrosine-based Activation Motif), chimeric molecules CD8-zeta, CD8-Igalpha, CD8-Igbeta, which contain the extracellular and transmembrane domains of the human CD8alpha molecule and the cytoplasmic tail of T-cell receptor (TcR) chain, Igalpha or Igbeta subunit of B-cell receptor, respectively, were stably expressed in a Jurkat cell line. Upon stimulation with anti-CD8 mAb OKT8, CD8-zeta and CD8-Igalpha chimeric proteins induced tyrosine phosphorylation of various cytoplasmic substrates as seen in TcR stimulation. They were also capable of stimulating IL-2 gene expression in a NF-AT dependent manner and inducing CD69 expression on the surface. However, stimulation of CD8-Igbeta can induce activation of CD69 surface expression and IL-2 gene expression equivalent to the level by CD8-Igalpha and CD8-zeta without induction of the tyrosine phosphorylation of intracellular signaling molecules. These results suggested that some of signaling chains containing ITAM may utilize a signal pathway without substrate tyrosine phosphorylation during T-cell activation leading to the IL-2 secretion.     

Alteration of lipid composition modulates Fc epsilon RI signaling in RBL-2H3 cells

We have used sonicated liposomes of phosphatidylcholine (PC), sphingomyelin (SM), or a mixture of cholesterol (chol) and PC to investigate the role of cellular lipid composition in Fc epsilon RI-mediated stimulation of RBL-2H3 cells. Overnight treatment with either PC or SM liposomes causes a substantial enhancement of antigen-stimulated degranulation and phospholipase A2 activity, whereas treatment with a PC/chol mixture results in partial inhibition of the antigen-stimulated response. The most consistent change in the cellular lipid composition that results from the PC and SM liposome treatments is an approximate 40% decrease in the chol/phospholipid (PL) ratio. The lipid treatments do not alter degranulation stimulated by AlF4- or by Ca2+ ionophore in the presence or absence of PMA, suggesting that lipid alteration affects a receptor-specific signaling process. The lipid treatments do not appear to alter antigen-stimulated tyrosine phosphorylation or Ca2+ mobilization. Possible involvement of protein kinase C (PKC) activation in the signal-enhancing effect of the PL treatments was investigated by using calphostin C and phorbol-12-myristol-13-acetate (PMA) to inhibit PKC activity and degranulation in RBL-2H3 cells. Both SM and PC treatment restore the antigen-mediated degranulation response that is inhibited by long-term treatment (> or = 16 h) with 100 nM PMA or short-term treatment (10 min) with 5 microM calphostin C. The results indicate that a decreased chol/PL ratio facilitates or enhances the receptor-mediated activation of a PKC-like pathway that plays an important role in Fc epsilon RI-stimulated degranulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential CD5-dependent regulation of CD5-associated CK2 activity in mature and immature T cells: implication on TCR/CD3-mediated activation

CD5 attenuates TCR-induced signals in immature thymocytes but functions as a costimulatory molecule potentiating TCR/CD3-mediated activation in mature, peripheral T cells. We have recently shown that the serine/threonine kinase, casein kinase 2 (CK2), a major regulator of cell growth and signaling, associates with and is activated by CD5. Therefore, we tested the possibility that differential regulation of CK2 activity by CD5 may be associated with these differences in CD5 modulation of TCR signaling. Consistent with our hypothesis, CD5-specific cross-linking activated associated CK2 in thymocytes but not active in mature splenocytes. Differential regulation of CD5-associated CK2 provides, for the first time, a potential mechanism for the differential effects of CD5 signaling in immature and mature cells. We propose that CD5 modulates Ag receptor activity through developmentally regulated activation of CD5-associated CK2.     

Reversal of human allergic T helper 2 responses by engagement of signaling lymphocytic activation molecule

Allergen-specific Th2 cells accumulate at high frequencies in the skin of patients with atopic dermatitis (AD), where they contribute to the induction and maintenance of the lesions that are characteristic for the disease. Attenuation of these lesions in response to successful therapy is associated with a reduction in IL-4-producing Th2 cells and the appearance of IFN-gamma-producing Th cells. In this study, we demonstrate that engagement of the signaling lymphocytic activation molecule (SLAM) by an agonistic mAb, during allergen-specific expansion of highly polarized Th2 cell populations derived from skin biopsies of AD patients, results in the generation of stable populations of IFN-gamma-producing cells. SLAM-mediated reversal of Th cell phenotype has important biologic consequences, because supernatants of these activated, allergen-specific Th cells fail to induce IgE synthesis by purified B cells costimulated by anti-CD40 mAbs. Thus, highly polarized, allergen-specific Th2 cell populations derived from the skin of AD patients can be reversed into Th cell populations that contain IFN-gamma-producing cells and that do not support IgE synthesis. These results define a new mechanism to promote Th0/Th1 differentiation and suggest a potential role for anti-SLAM mAbs in the treatment of Th2-mediated allergic diseases.