Modulation of choroidal neovascularization by subretinal injection of retinal pigment epithelium and polystyrene microbeads.

PURPOSE: The study was conducted to create a rapidly developing and reproducible animal model of subretinal choroidal neovascularization (CNV) that allows a time-dependent evaluation of growth dynamics, histopathologic features, and cytokine expression. METHODS: C57BL/6 and chemoattractant leukocyte protein-2 deficient (DeltaCcl-2) mice were studied. Mice received single or combined subretinal injections of cultured retinal pigment epithelium (RPE; C57BL/6-derived), polystyrene microbeads, or phosphate buffer solution (PBS). Fluorescence angiograms were performed over a period of 3 weeks. Mice were euthanized on post inoculation day 3, 7, 10, 14, or 21, and their eyes were evaluated by light, confocal, and electron microscopy. RESULTS: CNV membranes occurred in all study groups with an overall incidence of 94.3%. They extended in the subretinal space through central breaks in Bruch's membrane. CNV lesions were characterized by dynamic changes such as initiation, active inflammatory, and involution stages. CNV thickness peaked around PI day 7 and was greater in mice that received combined injections of RPE and microbeads or RPE cells alone. Small lesions developed in the control groups (microbeads or PBS only), in DeltaCcl-2, and old C57BL/6 mice. Variable expression of cytokines and growth factors was detected within the membranes. CONCLUSIONS: Our murine model represents a reliable approach inducing CNV growth by subretinal injection of either RPE cells alone or RPE cells and microbeads. The development of CNV lesions is a dynamic process that relies in part on macrophage trafficking and age.

Special regulatory T-cell review: Suppressors regulated but unsuppressed.

The rise-and-fall and reincarnation of suppressor T cells is reviewed from the perspective of a participant in the field.

CD8+ suppressor T cells resurrected.

This review focuses on the role of antigen-specific T cells that mediate active inhibition of immune responses over the past 35 years since their initial description. The field has experienced several changes in the accepted paradigm of such suppressor/regulatory T cells, from initial indications that such cells were CD8(+), to the view that such cells did not exist, to the identification of the transcription factor Foxp3 as a key orchestrator of inhibitory function. Although most Foxp3(+) cells in a resting animal are CD4(+)CD25(+) cells, Foxp3 expression and inhibitory function can be induced by antigens in the periphery by selective cytokine conditions, particularly TGF-beta. Such induced T cells occur within both the CD4 and the CD8 T-cell lineages and appear to mediate suppression by inhibiting the costimulatory activity of antigen-presenting cells and the production of inhibitory cytokines. Recent data generated by analysis of TCR Tg T cells that do not select many Foxp3-positive cells during thymic development are reviewed, emphasizing the pattern of "linked suppression" and focus of the relative potency of different mechanisms of suppression.

PI3 kinase enhancer-Homer complex couples mGluRI to PI3 kinase, preventing neuronal apoptosis.

Phosphoinositide 3 kinase enhancer (PIKE) is a recently identified nuclear GTPase that activates nuclear phosphoinositide 3-kinase (PI3 kinase). We have identified, cloned and characterized a new form of PIKE, designated PIKE-L, which, unlike the nuclear PIKE-S, localizes to both the cytoplasm and the nucleus. We demonstrate physiologic binding of PIKE-L to Homer, an adaptor protein known to link metabotropic glutamate receptors to multiple intracellular targets including the inositol 1,4,5-trisphosphate receptor (IP3R). We show that activation of group I metabotropic glutamate receptors (mGluRIs) enhances formation of an mGluRI-Homer-PIKE-L complex, leading to activation of PI3 kinase activity and prevention of neuronal apoptosis. Our findings indicate that this complex mediates the well-known ability of agonists of mGluRI to prevent neuronal apoptosis.

Ovalbumin serves as a neo-transplantation antigen in retinal pigment epithelial cells.

PURPOSE: Our long-term goal is to determine the optimal methods for inducing allograft tolerance to facilitate transplantation of retinal pigment epithelial cells or stem cells for the treatment of retinal degenerative diseases. These goals have been hampered by the extreme complexity of allograft rejection and the heterogeneity of responding T cells. The current studies were undertaken to develop a simplified transplant model for studying rejection and tolerance in the unique environment of the eye. METHODS: To provide a defined transplantation antigen, transgenic C57BL/6 (B6) mice were produced, which express the exogenous chicken egg ovalbumin (OVA) gene under the regulation of the mouse tyrosinase related protein-1 (TRP-1) promoter that is transcriptionally active in retinal pigmented epithelial (RPE) cells. To determine whether the transgene was expressed as a neo-transplantation antigen, RPE from TRP-1-OVA mice were injected into the subretinal space of B6 mice or B6 mice expressing the OVA-specific (OT1) TCR transgenes and examined for inflammatory cell infiltration. RESULTS: The TRP-1-OVA transgenic mice expressed OVA mRNA in the brain and eye but not the heart or kidney. RPE cells from TRP-1-OVA transgenic mice expressed mRNA and protein encoded by the OVA gene and RPE expressing TRP-1-OVA induced an inflammatory response within the subretinal space of OT1 mice but not in B6 mice. CONCLUSIONS: OVA serves as a defined, neo-transplantation antigen in RPE that is recognized by mice whose CD8+ T cells recognize OVA peptide. These observations provide new tools for future studies of the mechanisms of rejection and prolongation of RPE transplants in the eye.

Inhibition of cellular immune responses to encapsulated porcine islet xenografts by simultaneous blockade of two different costimulatory pathways.

BACKGROUND: Transplantation of human islets has been successful clinically. Since human islets are scarce, we are studying microencapsulated porcine islet xenografts in nonobese diabetic (NOD) mice. We have evaluated the cellular immune response in NOD mice with and without dual costimulatory blockade. METHODS: Alginate-poly-L-lysine-encapsulated adult porcine islets were transplanted i.p. in untreated diabetic NODs and NODs treated with CTLA4-Ig to block CD28/B7 and with anti-CD154 mAb to inhibit CD40/CD40-ligand interactions. Groups of mice were sacrificed on subsequent days; microcapsules were evaluated by histology; peritoneal cells were analyzed by FACS; and peritoneal cytokines were quantified by ELISA. Controls included immunoincompetent NOD-Scids and diabetic NODs given sham surgery or empty microcapsules. RESULTS: Within 20 days, encapsulated porcine islets induced accumulation of large numbers of macrophages, eosinophils, and significant numbers of CD4 and CD8 T cells at the graft site, and all grafts were rejected. During rejection, IFNgamma, IL-12 and IL-5 were significantly elevated over sham-operated controls, whereas IL-2, TNFalpha, IL-4, IL-6, IL-10, IL-1beta and TGFbeta were unchanged. Treatment with CTLA4-Ig and anti-CD154 prevented graft destruction in all animals during the 26 days of the experiment, dramatically inhibited recruitment of host inflammatory cells, and inhibited peritoneal IFNgamma and IL-5 concentrations while delaying IL-12 production. CONCLUSIONS: When two different pathways of T cell costimulation were blocked, T cell-dependent inflammatory responses were inhibited, and survival of encapsulated islet xenografts was significantly prolonged. These findings suggest synergy between encapsulation of donor islets and simultaneous blockade of two host costimulatory pathways in prolonging xenoislet transplant survival.

Regulation of CD8+ cytolytic T lymphocyte differentiation by a cholinergic pathway.

In this report, we provide evidence that muscarinic receptors play a role in the generation of CD8+ cytolytic T lymphocytes. Analysis of mice with targeted deletions of each of the known muscarinic receptors (M1-M5) showed that CD8+ T cells from M1 receptor-deficient mice had a defect in the ability to differentiate into cytolytic T lymphocytes. Additional pharmacological experiments support the role of muscarinic receptors in wild type mice and suggest that acetylcholine may be involved. Together, these findings suggest that the M1 muscarinic receptor is involved in CTL development, thus providing novel insights into CD8+ T cell biology and the potential role of cholinergic signaling in immune regulation.

Use of superparamagnetic microbeads in tracking subretinal injections.

PURPOSE: The purpose of these studies was to develop a method to track intraocular injections. METHODS: Retinal pigment epithelial (RPE) cells, purified from adult mouse eyes, were incubated with superparamagnetic microbeads (Dynabeads, 4.5 microm) coated with bovine serum albumin to verify that they could phagocytose the microbeads. For in vivo tracking studies, mice were anesthetized and a small incision was made at the pars plana and 2 mul of microbeads (around 10(5) microbeads) or RPE that had taken up the microbeads were injected into the subretinal space (SRS). Mice were sacrificed at various times after injection. The eyes were enucleated, fixed in formalin, and embedded in paraffin. Sections were stained with H&E, visualized by light microscopy. Some eyes were digested with collagenase and inflammatory cells determined by flow cytometry. RESULTS: Cultured adult RPE phagocytosed the magnetic microbeads. One day after injection into the SRS, a retinal detachment was observed at the injection point and free microbeads were easily detected at this site. One week later, the host RPE cells had phagocytized the microbeads and the retina had reattached. No inflammatory response was detected in the eyes that were injected with microbeads in the SRS at any time examined. Histology showed normal morphology of all retinal layers around the injection site. The microbeads remained in situ throughout the study. CONCLUSIONS: Protein coated magnetic microbeads are non-inflammatory after injection into the SRS. Host RPE cells phagocytized the microbeads and the retina maintained a healthy morphology after reattachment. This technique proved not only to be a good training tool to determine the precise location of injection, but also provided a noninflammatory method for long term marking of delivery into the SRS. However, the microbeads can not be used as a tracer of injected RPE because the microbeads were readily transferred to the endogenous RPE.

CD8+ T-cell tolerance induced by delivery of antigen to the anterior chamber is not the result of de facto intravenous or mucosal administration of antigen.

PURPOSE: We tested whether antigen administration via the anterior chamber (a.c.) was equivalent to intravenous (i.v.) or mucosal administration antigen. METHODS: Ovalbumin (OVA)-specific CD8(+) T cells (OT-I) were enumerated in lymphoid tissues of C57Bl/6 (B6) mice via adoptive transfer after the same amount of antigen was administered via a.c., i.v., or mucosal routes. Lytic activity was measured in B6 and gammadeltaT cell-deficient B6 mice given OVA via a.c., i.v, or mucosal routes after injection with OVA in adjuvant. RESULTS: OVA a.c. induced a pattern of T-cell proliferation distinct from i.v. or mucosal administration. A.c. and i.v., but not mucosal, OVA induced cytolytic T lymphocyte (CTL) tolerance. The inhibition of CTL responses was significantly greater in mice given OVA a.c. rather than i.v. gammadeltaT cells contributed to a.c.-, but not i.v.-, induced CTL tolerance. CONCLUSIONS: A.c. administration of antigen not de-facto i.v. or mucosal administration of antigen.

Ocular immune privilege and CTL tolerance.

The introduction of antigens into the anterior chamber (AC) of the eye, an immune-privileged site, induces immune responses that effectively eliminate ocular pathogens while minimizing tissue damage that can cause blindness. This specialized immune response, termed AC associated immune deviation (ACAID) is thought to be an evolutionary compromise to preserve the delicate microanatomy of the eye while maintaining ocular immune responses. The injection of soluble antigen in the AC of mice results in systemic tolerance characterized by reduced priming for antigen-specific delayed-type hypersensitivity (DTH) and cytotoxic T lymphocyte (CTL) responses. Similarly, the injection of histo incompatible tumors into the AC of mice reduces priming for DTH responses specific to minor antigens. However, robust tumor-specific CTL responses are induced systemically following this treatment that are capable of eliminating a subsequent injection of the same tumors in the skin or the opposite eye. Interestingly, CTL responses induced by administration of tumors in the AC fail to eliminate the primary ocular tumor. In this review, we compare and contrast CTL responses generated by the injection of soluble or tumor-associated antigens in the AC and discuss mechanisms employed to induce ocular CTL tolerance.

Identification and characterization of CD8+ suppressor T cells.

It has long been appreciated that certain subsets of T cells are capable of suppressing immune reactions. Initially, such T cells were described as CD8+ suppressor T cells (Ts) and there is a vast body of research spanning 30 years that describes the immunobiology of CD8+ Ts. However, studies on CD8+ Ts have suffered from the inability to distinguish CD8+ Ts from CD8+ T cells of other phenotypes. Here we present a brief history of CD8+ Ts, a review of recent progress distinguishing CD8+ Ts as a unique subset of CD8+ T cells, and an overview of the evolving immunological context in which CD8+ Ts function.

Gammadelta T cells play an essential role in several forms of tolerance.

Gammadelta T cells were discovered in the mid-1980s, but the antigens they recognize and the biological functions they mediate are poorly understood. Although gammadelta T cells have the capacity to augment immunity to certain infections and kill certain tumor cells, they are generally not required for development of antibody responses, for graft rejection, or for development of autoimmune diseases. Nevertheless, gammadelta T cells accumulate at sites of inflammation induced by infection, tumor growth, and autoimmune lesions, where they have been shown to reduce the inflammatory reaction and tissue damage. In this review, we summarize the evidence that gammadelta T cells play an important role in the induction of various forms of tolerance.

Gammadelta T cells in anterior chamber-induced tolerance in CD8(+) CTL responses.

PURPOSE: Delivery of antigen to the anterior chamber (AC) of the eye induces a systemic form of tolerance, referred to as anterior chamber-associated immune deviation (ACAID). ACAID is characterized by decreases in delayed-type hypersensitivity responses and complement-fixing antibodies on subsequent challenge with an immunogenic form of the antigen. The current study was designed to test whether priming of antigen-specific CD8(+) cytotoxic T-lymphocytes (CTLs) are inhibited by injection of soluble antigen into the AC and whether gammadelta T cells play a role in the inhibition of such responses. METHODS: Antigen was administered through the AC to normal gammadelta T-cell-deficient or reconstituted gammadelta T-cell-deficient mice. Seven days after the AC injection, the mice were primed with antigen in adjuvant and 10 days later, their spleen cells were cultured for 5 to 7 days and the CTL responses measured. RESULTS: CTL responses were inhibited by antigen delivered through the AC in normal but not gammadelta T-cell-deficient mice. Tolerance was reconstituted in delta-chain knockout mice by the adoptive transfer of gammadelta T cells from normal mice. Moreover, spleen cells and splenic gammadelta(+) T cells, but not gammadelta(-) T cells, from mice injected with antigen through the AC inhibited development of CTL responses when cultured together with primed effector T cells. CONCLUSIONS: These data show, for the first time, that administration of soluble antigen in the AC inhibits development of CD8(+) cytotoxic T-cell responses and that gammadelta T cells play a critical role in inhibition of CTL responses.

Proliferative and cytokine responses in CTLA4-Ig-treated diabetic NOD mice transplanted with microencapsulated neonatal porcine ICCs.

Our goal is to develop effective islet xenografts for treating human diabetes. We have studied microencapsulated neonatal porcine islet cell clusters (ICCs) transplanted intraperitoneally in spontaneously diabetic NOD mice, where they function to maintain normoglycemia in the autoimmune host. Nonencapsulated neonatal porcine ICCs functioned for 4.5 +/- 0.5 days before being rejected; encapsulation prolonged graft function to 17 +/- 2 days. CTLA4-Ig treatment did not enhance the survival of nonencapsulated ICCs. However, CTLA4-Ig treatment significantly extended the function of encapsulated ICCs to 73 +/- 5 days. Histological analyses demonstrated a profuse pericapsular cellular reaction associated with rejection of encapsulated islet xenografts in untreated mice, while this reaction was significantly reduced in CTLA4-Ig-treated mice. To study mechanisms of xenograft rejection in this model, we analyzed proliferative responses to neonatal porcine ICCs and cytokines present in the peritoneal cavities of transplanted mice. Spleen cells from both CTLA4-Ig-treated and untreated rejecting NODs exhibited vigorous proliferation in the absence of antigenic stimulation, suggesting prior activation in vivo, while splenocytes from CTLA4-Ig-treated NODs with functioning grafts had low proliferative levels, equal to controls. Islet-specific proliferation was not detected in islet-rejecting mice, perhaps due to their high background levels. With the exception of elevated IL-6 levels, empty capsules did not provoke a significant peritoneal cytokine response compared with sham surgery or untransplanted control mice. However, IL-5, IL-12, TGF-beta, and IL-1beta were significantly elevated in NODs receiving encapsulated neonatal porcine ICCs compared with untransplanted controls. There were no significant differences between peritoneal cytokine concentrations in CTLA4-Ig-treated mice with long-term functioning grafts compared to mice that rejected grafts at earlier time points. We conclude that the combination of donor islet microencapsulation and brief treatment of the recipient with co-stimulatory blockade delays sensitization of the host, possibly by altering mechanism(s) for recruitment and/or activation of host effector cells.

Antigen, in the presence of TGF-beta, induces up-regulation of FoxP3gfp+ in CD4+ TCR transgenic T cells that mediate linked suppression of CD8+ T cell responses.

CD4(+)CD25(+) regulatory T cells (Tregs) inhibit immune responses to a variety of Ags, but their specificity and mechanism of suppression are controversial. This controversy is largely because many studies focused on natural Tregs with undefined specificities and suppression has frequently been measured on polyclonal T cell responses. To address the issue of specificity further, we have bred K(d)-specific, CD4(+) TCR (TCR75) transgenic mice to Foxp3(gfp) knockin reporter mice to permit sorting of Tregs with a known specificity. Foxp3(gfp).TCR75 mice did not express significant numbers of natural FoxP3(+) Tregs expressing the TCR75 transgenes, but FoxP3 expression was induced by stimulating with K(d) plus TGF-beta. The resulting GFP(+) TCR75 cells were anergic, whereas the GFP(-) TCR75 cells proliferated upon restimulation with K(d) peptide. Yet both exhibited severely reduced expression of intracellular IFN-gamma and TNF-alpha upon restimulation. GFP(+), but not GFP(-), TCR75 T cells suppressed responses by naive TCR75 T cells and by nontransgenic spleen cells stimulated with anti-CD3. GFP(+) TCR75 cells also inhibited polyclonal C57BL/6 anti-K(d) CTL responses if the APC expressed K(d) and both MHC class I and class II, and responses by OT1 T cells to B6.K(d).OVA but not B6.K(d) plus OVA expressing APC, demonstrating linked-suppression of CD8 responses. Thus, Tregs exhibit a greater degree of specificity in vitro than previously appreciated. The observation that Tregs and responder T cells must recognize the same APC provides a mechanistic explanation for the observation that Tregs must be in direct contact with effector T cells to suppress their responses.

Accumulation of immunosuppressive CD11b+ myeloid cells correlates with the failure to prevent tumor growth in the anterior chamber of the eye.

The purpose of these studies is to determine why an immunogenic tumor grows unchecked in the anterior chamber (a.c.) of the eye. The OVA-expressing EL4 tumor, E.G7-OVA, was injected into the a.c. or skin of immunocompetent and immunodeficient mice. Tumor growth and tumor-specific immune responses were monitored. Ocular tumor-infiltrating leukocytes were characterized phenotypically and functionally. Growth of E.G7-OVA was inhibited when limiting numbers of cells were injected in the skin but not in the a.c. of C57BL/6 mice, although both routes primed OVA-specific immune responses, which prevented the growth of a subsequent injection with E.G7-OVA in the skin or opposite eye. Tumor regression was OVA-specific because growth of the parental EL-4 tumor was not inhibited in primed mice. E.G7-OVA growth in the skin was not inhibited in immunodeficient Rag(-/-) or CD8 T cell-deficient mice, suggesting that CD8(+) CTLs mediate tumor elimination. CD8(+) T cell numbers were significantly increased in eyes of mice primed with E.G7-OVA, but few were detected in primary ocular tumors. Nevertheless, growth of E.G7-OVA was retarded in the a.c. of TCR-transgenic OT-I mice, and CD8(+) T cell numbers were increased within eyes, suggesting that tumor-specific CD8(+) CTLs migrated into and controlled primary ocular tumor growth. E.G7-OVA did not lose antigenicity or become immunosuppressive after 13 days of growth in the eye. However, CD11b(+) cells accumulated in primary ocular tumors and contained potent immunosuppressive activity when assayed in vitro. Thus, CD11b(+) cells that accumulate within the eye as tumors develop in the a.c. may contribute to immune evasion by primary ocular tumors by inhibiting CTLs within the eye.

TCR transgenic CD8+ T cells activated in the presence of TGFbeta express FoxP3 and mediate linked suppression of primary immune responses and cardiac allograft rejection.

Although CD4+CD25+FoxP3+ regulatory T cells play a role in allograft tolerance, the role of CD8+ cells with immunosuppressive function is less clear. To address this issue, spleen cells from Rag-1-deficient TCR transgenic (Tg) mice expressing a receptor for ovalbumin (OVA) in the context of MHC class I (OT1) were activated with OVA expressing antigen-presenting cell (APC) in the presence or absence of exogenous transforming growth factor beta (TGFbeta). TGFbeta inhibited the expression of IFN-gamma, granzyme B and the lytic activity of the OT1 T cells while inducing FoxP3 expression in 5-15% of the cells. By contrast, FoxP3 expression was not detected in naive OT-1 T cells or OT-1 T cells activated without exogenous TGFbeta. TGFbeta-activated OT1 cells inhibited the activation of Kd-specific CD8+ CTL responses by normal B6 T cells and the proliferation by Kd-specific CD4+ TCR Tg T cells, but only if the OVA epitope was co-expressed by Kd+ APC. This antigen-specific inhibitory activity, referred to as linked suppression, was neither mediated by residual lytic activity within the activated OT1 T cells nor did it depend upon IL-10 or TGFbeta. Suppression correlated with inhibition of CD86 expression on CD11c+ APC. TGFbeta-activated OT1 T cells also delayed the rejection of heterotopic, vascularized cardiac allografts mediated by anti-Kd-specific CD4+ TCR Tg T cells, but only if the cardiac allograft expressed both OVA and Kd as transgenes. Prolonged survival of allografts was associated with rapid migration of the FoxP3+ OT1 T cells into the donor heart raising the possibility that suppression may be mediated within the allograft. These data show that TGFbeta-activated CD8+ T cells mediate antigen-specific, APC-focused patterns of suppression in vitro and in vivo.

Inhibition of tumor rejection by gammadelta T cells and IL-10.

Although many tumors express tumor-specific antigens, most fail to stimulate effective immune responses. Tumors generally lack co-stimulatory molecules, which can lead to tolerance of tumor-specific T cells and progressive tumor growth. Here, we demonstrate that the ovalbumin (OVA) transfected EL4 tumor, E.G7-OVA, grows progressively in syngeneic mice even though the tumor can be rejected if the mice are immunized with OVA in adjuvant. E.G7-OVA grew more rapidly in RAG-1 deficient than sufficient mice suggesting that normal mice make an abortive immune response to this tumor. Depletion of gammadelta T cells or IL-10 augmented the ability of B6 mice to reject E.G7-OVA. Spleen cells from normal, but not IL-10 knockout, mice reconstituted rapid tumor growth in gammadelta T cell-deficient mice. Thus, gammadelta T cells play an important role in preventing immune elimination of this tumor by a mechanism that directly or indirectly involves IL-10.

Injection of soluble antigen into the anterior chamber of the eye induces expansion and functional unresponsiveness of antigen-specific CD8+ T cells.

The injection of soluble Ag into the anterior chamber (a.c.) of the eye induces systemic tolerance, termed a.c.-associated immune deviation (ACAID), characterized by Ag-specific inhibition of delayed-type hypersensitivity responses and a reduction in complement-fixing Abs. Recently, we have shown that CD8(+) CTL responses are also inhibited in ACAID. In this study, we have used an adoptive transfer approach to follow the fate of Ag-specific CD8(+) TCR transgenic (OT-I) T cells in vivo during the induction and expression of ACAID. C57BL/6 (B6) recipients of OT-I splenocytes that were injected with chicken OVA in the a.c. displayed reduced OVA-specific delayed-type hypersensitivity and CTL responses, compared with those of mice given OVA in the subconjunctiva or an irrelevant Ag human IgG in the a.c. OT-I T cells increased 9-fold in the submandibular lymph nodes and 3-fold in the spleen following an a.c. injection with OVA, indicating that expansion rather than deletion of Ag-specific CD8(+) T cells was induced by this treatment. OT-I T cells expanded equivalently upon administration of OVA in CFA to mice previously given OVA in the a.c. or subconjunctiva. However, the lytic activity attributed to OT-I T cells was reduced on a per-cell basis in mice previously given OVA in the a.c. We conclude that tolerance of CTL responses in mice given Ag via the a.c. results from unresponsiveness of Ag-specific CD8(+) T cells.

Secretion of IL-2 and IFN-gamma, but not IL-4, by antigen-specific T cells requires extracellular ATP.

Extracellular ATP and other nucleotides transmit signals to cells via surface-associated molecules whose binding sites face the extracellular milieu. Ecto-nucleoside triphosphate diphosphohydrolase is such an ATP-binding enzyme that is expressed by activated lymphocytes. We have previously shown that nonhydrolyzable ATP analogs block the lytic activity of NK cells and CD8(+) T cells as well as their E-NTPDase activity. These results suggest that the hydrolysis of ATP may play a role in lymphocyte function. Here we report that E-NTPDase activity is up-regulated within 15 min of T cell stimulation and that reversible and irreversible enzyme inhibitors profoundly reduce secretion of IL-2 and IFN-gamma, but not IL-4. TNF-alpha, IL-10, and IL-5 production showed intermediate sensitivity to these ATP analogs. Depletion of extracellular ATP also inhibited secretion of IFN-gamma, but not IL-4, supporting the interpretation that extracellular ATP is required for secretion of some, but not all, cytokines. E-NTPDase antagonists reduced transcription of IL-2 mRNA and inhibited TCR-mediated intracellular calcium flux. These results suggest that extracellular ATP plays an essential role in the TCR-mediated signal transduction cascade for expression of certain cytokine genes.