Sergey Apasov - Academia.edu (original) (raw)

Papers by Sergey Apasov

Research paper thumbnail of Highly Lytic CD8+, T-Cell Receptor Cytotoxic T Cells with Major Histocompatibility Complex (MHC) Class I Antigen-Directed Cytotoxicity in 2-Microglobulin, MHC Class I-Defiencient Mice

Research paper thumbnail of Generation of Antigen-Specific Murine CTL Under Weakly Immunogenic Conditions

Cytotoxic Cells: Recognition, Effector Function, Generation, and Methods, 1993

Research paper thumbnail of Multidrug-resistance phenotype of a subpopulation of T-lymphocytes without drug selection

Experimental Cell Research, 1989

Research paper thumbnail of A2A receptor dependent and A2A receptor independent effects of extracellular adenosine on murine thymocytes in conditions of adenosine deaminase deficiency

Blood, Jun 15, 2000

Adenosine deaminase (ADA) deficiency causes severe combined immunodeficiency (SCID) and is accomp... more Adenosine deaminase (ADA) deficiency causes severe combined immunodeficiency (SCID) and is accompanied by T-cell depletion and accumulation of both intracellular and extracellular adenosine (extAdo) and deoxyadenosine. To better understand the causes of T-cell depletion in vivo and to discriminate between extracellular and intracellular effects of exogenously added adenosine in vitro, we investigated mechanisms of 2 different effects of adenosine on murine thymocytes. These effects of adenosine include direct induction of apoptosis in about 6% to 15% thymocytes and inhibition of T-cell receptor TCR)-induced activation of the majority of thymocytes with inhibited ADA. A 2A adenosine receptors, but not A 2B , A 1 , or A 3 receptors, are shown to be mostly responsible for extAdo-triggered signaling (cyclic adenosine monophosphate [cAMP] accumulation) in murine thymocytes and this prompted studies of the effects of extAdo on thymocytes from A 2A R gene-deficient mice. It is found that direct apoptotic effects of extAdo on CD4 ؉ CD8 ؉ double positive (DP) thymocytes are completely accounted for by signaling through A 2A R, with no contribution of intracellular lymphotoxicity or of compensating A 2B Rs because only A 2A R ؉/؉, but not A 2A R ؊/؊ thymocytes were susceptible to apoptotic effects of ex-tAdo. Studies of the effects of cAMPraising agents support observations of extAdo/A 2A R/cAMP-triggered apoptosis in DP thymocytes. Unexpectedly, the extAdo strongly inhibited TCR-triggered activation of both A 2A R ؉/؉ and A 2A R ؊/؊ thymocytes in the presence of ADA inhibitors. This was confirmed with thymocytes from ADA gene-deficient mice, suggesting the existence of A 2A R-independent effects of extAdo on thymocytes. The presented data raises questions about the identity and functional role of A 2A R-expressing thymocytes in T-cell differentiation and of the role of TCRantagonizing effects of extAdo in conditions of ADA SCID. (Blood. 2000;95: 3859-3867)

Research paper thumbnail of Highly Lytic CD8^+, alphabeta T-Cell Receptor Cytotoxic T Cells with Major Histocompatibility Complex (MHC) Class I Antigen-Directed Cytotoxicity in beta_2-Microglobulin, MHC Class I-Defiencient Mice

Pnas, 1993

Targeted disruption of the beta 2-microglobulin (beta 2m) gene results in major histocompatibilit... more Targeted disruption of the beta 2-microglobulin (beta 2m) gene results in major histocompatibility complex (MHC) class I deficiency and virtual disappearance of functional CD8+ cytotoxic T lymphocytes (CTLs) in beta 2m-deficient (beta 2m-/-) mice. We asked whether the beta 2m-/- mice are able to reject tumor cells injected i.p. and what is the cellular composition of peritoneal exudate leukocytes (PELs) from such mice. We found that beta 2m-/- mice do reject MHC class I-bearing tumor cells injected i.p. Surprisingly, analysis of PEL CTLs obtained from i.p. tumor-injected beta 2m -/- mice revealed the presence of a large proportion of functional, tumor-destroying CD8+, CD4-, alpha beta T-cell receptor-positive, CD3+, Thy-1+, MHC class I-negative CTLs with strong MHC class I-directed cytotoxic activity. These results call for careful studies of local accumulation of CD8+ CTLs in beta 2m -/- mouse models and suggest that the dramatic decrease in MHC class I expression caused by beta 2m gene disruption does not prevent CD8+/CD4- cell selection and expansion.

Research paper thumbnail of Development and antigen specificity of CD8+ cytotoxic T lymphocytes in β2-microglobulin-negative, MHC class I-deficient mice in response to immunization with tumor cells

The Journal of Immunology, 1994

beta 2-Microglobulin knockout mice (beta 2-m-/-) with MHC class I expression deficiency are able ... more beta 2-Microglobulin knockout mice (beta 2-m-/-) with MHC class I expression deficiency are able to develop functional TCR(+)-alpha beta, CD8+ CTLs in response to tumor cell injection. The i.p. injection of beta 2-m-/- mice with tumor results in the massive accumulation of highly lytic CD8+ CTLs in the peritoneum and causes the local recruitment of CD8+ T cells into lymph nodes and spleens of immune animals. The accumulation of CD8+ CTLs in peritoneum is accompanied by the rejection of tumor cells and the survival of animals. The deficiency in MHC class I expression in beta 2-m/- mice is reflected in the delayed tumor rejection and CD8+ cell accumulation during the primary anti-tumor response in comparison with normal mice. The secondary response, however, is identical in normal and MHC class I-deficient mice. The rejection of tumor cells appears to be MHC class I directed because no rejection of tumors, no accumulation of CD8+ CTLs, and no survival of animals were observed when syngeneic tumor cells were used for injection with the notable exception of anti-minor Ag response. The Ag specificity of CD8+ CTLs in beta 2-m-/- mice is demonstrated using a panel of tumor target cells and class I transfectants. Although no substantial differences were found in the number and specificity of peritoneal CD8+ CTLs in beta 2-m-/- and normal mice using tumor rejection studies, the analysis of TCR-V beta phenotype using the panel of mAbs revealed the reduction in proportion of TCR-V beta 5 and TCR-V beta 6 used by CD8+ cell population from beta 2-m-/- mice. Development of lytic and H-2-directed CD8+ cells in regional lymph nodes was also observed after footpad immunization of beta 2-m-/- mice with TNP-labeled C57BL/6 splenocytes, suggesting anti-minor Ag reaction.

Research paper thumbnail of Critical Role of Hypoxia and A2A Adenosine Receptors in Liver Tissue-Protecting Physiological Anti-Inflammatory Pathway

Research paper thumbnail of Analysis of A2a receptor-deficient mice reveals no significant compensatory increases in the expression of A2b, Al, and A3 adenosine receptors in lymphoid organs

Biochemical Pharmacology, 2003

Although recent genetic and pharmacologic in vivo studies of acute inflammation models in mice de... more Although recent genetic and pharmacologic in vivo studies of acute inflammation models in mice demonstrated that the cyclic AMP-elevating A2a receptor plays a non-redundant role in protection from excessive acute inflammatory tissue damage and in the down-regulation of proinflammatory cytokine production, it remained to be established whether genetic deficiency of the A2a receptor is accompanied by a compensatory up-regulation of the cAMP-elevating A2b receptor and/or other adenosine receptors. Here, we show that most of the cAMP response to adenosine is abolished in lymphoid tissues of A2a receptor-deficient mice, although some response remains in splenocytes. No significant changes were observed in A2b, A1, and A3 mRNA levels in the thymus or lymph nodes of A2a receptor-deficient mice, but small increases in mRNA expression of these receptors were detected in the spleen. These data suggest that regulation of the expression of A2b, A1, and A3 receptors is not affected significantly by the absence of A2a receptors and may provide further explanation of earlier in vivo observations of increased tissue damage and of longer persistence of proinflammatory cytokines in animals with inactivated A2a receptors.

Research paper thumbnail of Role of A2a extracellular adenosine receptor-mediated signaling in adenosine-mediated inhibition of T-cell activation and expansion

Blood, Aug 15, 1997

(2) the A2a, but not the A1 or A3, receptors are the major absence of adenosine deaminase activit... more (2) the A2a, but not the A1 or A3, receptors are the major absence of adenosine deaminase activity (ADA) activity results in lymphocyte depletion and in severe combined im-expressed and functionally coupled adenosine receptors in mouse peripheral T and B lymphocytes, and the adenosine-munodeficiency (ADA SCID), which is currently explained by direct cell death-causing effects of intracellular products of induced cAMP accumulation in lymphocytes correlates with the expression of A2a receptors; (3) the specific agonist of adenosine metabolism. We explored the alternative mechanisms of peripheral T-cell depletion as due to inhibition of A2a receptor, CGS21680, induces increases in [cAMP]i in lymphocytes, whereas the specific antagonist of A2a recep-T-cell expansion by extracellular adenosine-mediated signaling through purinergic receptors. The strong inhibition of tor, CSC, inhibits the effects of Ado and CGS21680; and (4) the increases in [cAMP]i mimic the adenosine-induced inhi-the T-cell receptor (TCR)-triggered proliferation and of upregulation of interleukin-2 receptor a chain (CD25) mole-bition of TCR-triggered CD25 upregulation and splenocyte proliferation. These studies suggest the possible role of cules, but not the direct lymphotoxicity, were observed at low concentrations of extracellular adenosine. These effects adenosine receptors in the regulation of lymphocyte expansion and point to the downregulation of A2a purinergic re-of extracellular adenosine (Ado) are likely to be mediated by A2a receptor-mediated signaling rather than by intracellular ceptors on T cells as a potentially attractive pharmacologic target. toxicity of adenosine catabolites, because (1) poorly metabolized adenosine analogs cause the accumulation of cAMP ᭧ tively low concentrations of adenosine rather then the direct,

Research paper thumbnail of Studies of expression and possible functional role of purinergic receptors in cell-mediated immunity: Experimental approaches, controls, and caveats

Drug Develop Res, 1998

ABSTRACT

Research paper thumbnail of Purinoreceptors and T Cell Function: Regulation of Lymphocyte Activation by Extracellular ATP and Adenosine

Developments in Cardiovascular Medicine, 1998

Research paper thumbnail of Identification of Protein Kinases and Protein Phosphatases Involved in CTL Effector Functions. “ON” and “OFF” Signaling and Immunopharmacological Implications

Cytotoxic Cells: Recognition, Effector Function, Generation, and Methods, 1993

Research paper thumbnail of Critical role of hypoxia and A2A adenosine receptors in liver tissue-protecting physiological anti-inflammatory pathway

Molecular medicine (Cambridge, Mass.)

Whole body exposure of wild type control littermates and A2A adenosine receptor (A2AR) gene delet... more Whole body exposure of wild type control littermates and A2A adenosine receptor (A2AR) gene deleted mice to low oxygen containing inspired gas mixture allowed the investigation of the mechanism that controls inflammatory liver damage and protects the liver using a mouse model of T cell-mediated viral and autoimmune hepatitis. We tested the hypothesis that the inflammatory tissue damage-associated hypoxia and extracellular adenosine --> A2AR signaling plays an important role in the physiological anti-inflammatory mechanism that limits liver damage during fulminant hepatitis. After induction of T cell-mediated hepatitis, mice were kept in modular chambers either under normoxic (21% oxygen) or hypoxic (10% oxygen) conditions for 8 h. It was shown that the whole body exposure to hypoxic atmosphere caused tissue hypoxia in healthy animals as evidenced by a decrease in the arterial blood oxygen tension and increase of the plasma adenosine concentration (P < 0.05). This "hypoxic...

Research paper thumbnail of T cell-mediated immunity

Principles of Immunopharmacology, 2005

Research paper thumbnail of A2A adenosine receptor protects tumors from antitumor T cells

Proceedings of the National Academy of Sciences, 2006

The A2A adenosine receptor (A2AR) has been shown to be a critical and nonredundant negative regul... more The A2A adenosine receptor (A2AR) has been shown to be a critical and nonredundant negative regulator of immune cells in protecting normal tissues from inflammatory damage. We hypothesized that A2AR also protects cancerous tissues by inhibiting incoming antitumor T lymphocytes. Here we confirm this hypothesis by showing that genetic deletion of A2AR in the host resulted in rejection of established immunogenic tumors in Ϸ60% of A2ARdeficient mice with no rejection observed in control WT mice.

Research paper thumbnail of Transient up-regulation of P2Y2 nucleotide receptor mRNA expression is an immediate early gene response in activated thymocytes

Proceedings of the National Academy of Sciences, 1997

In studies designed to understand the roles of P2 nucleotide receptors in differentiation of T ly... more In studies designed to understand the roles of P2 nucleotide receptors in differentiation of T lymphocytes, we observed a transient and protein synthesis-independent enhancement of mRNA expression for the G protein-coupled P2Y 2 receptor in mouse thymocytes after the addition of steroid hormone or T cell receptor (TCR) crosslinking by anti-TCR mAb. Conversely, dexamethasone-induced increases in mRNA expression for the ligand-gated ion channel P2X 1 receptor was detected in rat, but not mouse, thymocytes, raising questions about the previously suggested role of P2X 1 receptors in thymocyte apoptosis. Flow cytometry analysis of thymocyte subsets excluded the possibility that the observed increases in P2Y 2 receptor mRNA expression were due to the enrichment of steroid-treated cells with an P2Y 2 mRNA-rich thymocyte subset. Triggering of TCR-mediated intracellular signaling pathways through crosslinking of TCR or by addition of phorbol ester and Ca 2؉ ionophore also resulted in the up-regulation of P2Y 2 , but not P2X 1 , receptor mRNA. It is proposed that the rapid increase of P2Y 2 receptor mRNA expression could be a common early event in responses of T cells to different activating stimuli. Taken together with the recently discovered ability of nucleotide receptor-initiated signaling to antagonize or enhance the effects of TCR crosslinking or steroids on thymocytes, the observed rapid up-regulation of P2Y 2 receptor mRNA expression may reflect an immediate early gene response where newly expressed cell surface nucleotide receptors provide regulatory feedback signaling from extracellular ATP in the T cell differentiation process.

Research paper thumbnail of A Serine/Threonine Phosphorylation Site in the Ectodomain of a T Cell Receptor β Chain is Required for Activation by Superantigen

Journal of Receptors and Signal Transduction, 2003

The presence of consensus phosphorylation sites in the ectodomains of cell surface proteins sugge... more The presence of consensus phosphorylation sites in the ectodomains of cell surface proteins suggests that such post-translational modification may be important in regulation of surface receptor activity. To date, the only cell surface receptor for which such ectodomain phosphorylation has been conclusively demonstrated is the clonally expressed T cell antigen receptor (TCR). Attempts to conclusively identify individual phosphorylated residues in TCR alpha and beta chains and determine their functional significance by biochemical approaches failed due to insufficient quantities of purified molecules. Here we present the results of an alternative approach where survey of phosphorylation sites in the TCR alpha and beta chains was accomplished using site-directed mutagenesis and retroviral vector expression, as well as in vitro phosphorylation of synthetic peptide substrates. All mutants studied directed the cell surface expression of normal amounts of TCR, and all transfectants could be stimulated to produce IL-2 in response to substrate-immobilized antibody to TCR. However, mutation of serine-88 in the protein kinase A phosphorylation site of the TCR beta chain resulted in a complete lack of response to the superantigen staphylococcal enterotoxin B (SEB). In addition, this mutation abolished TCR-associated tyrosine phosphorylation, consistent with the impairment of cell signaling. Reversion of the serine-88/alanine mutation with phosphorylatable threonine completely restored the SEB recognition by TCR. These results, interpreted in the context of the known three-dimensional structure of the complex of SEB and TCR, are consistent with the view that serine-88 is important for the contact of the TCR beta chain with SEB.

Research paper thumbnail of Cutting Edge: Physiologic Attenuation of Proinflammatory Transcription by the Gs Protein-Coupled A2A Adenosine Receptor In Vivo

The Journal of Immunology, 2004

Research paper thumbnail of Differential Effects of Physiologically Relevant Hypoxic Conditions on T Lymphocyte Development and Effector Functions

The Journal of Immunology, 2001

Direct measurements revealed low oxygen tensions (0.5-4.5% oxygen) in murine lymphoid organs in v... more Direct measurements revealed low oxygen tensions (0.5-4.5% oxygen) in murine lymphoid organs in vivo. To test whether adaptation to changes in oxygen tension may have an effect on lymphocyte functions, T cell differentiation and functions at varying oxygen tensions were studied. These studies show: 1) differentiated CTL deliver Fas ligand- and perforin-dependent lethal hit equally well at all redox conditions; 2) CTL development is delayed at 2.5% oxygen as compared with 20% oxygen. Remarkably, development of CTL at 2.5% oxygen is more sustained and the CTL much more lytic; and 3) hypoxic exposure and TCR-mediated activation are additive in enhancing levels of hypoxia response element-containing gene products in lymphocyte supernatants. In contrast, hypoxia inhibited the accumulation of nonhypoxia response element-containing gene products (e.g., IL-2 and IFN-gamma) in the same cultures. This suggests that T cell activation in hypoxic conditions in vivo may lead to different patterns of lymphokine secretion and accumulation of cytokines (e.g., vascular endothelial growth factor) affecting endothelial cells and vascular permeabilization. Thus, although higher numbers of cells survive and are activated during 20% oxygen incubation in vitro, the CTL which develop at 2.5% oxygen are more lytic with higher levels of activation markers. It is concluded that the ambient 20% oxygen tension (plus 2-ME) is remarkably well suited for immunologic specificity and cytotoxicity studies, but oxygen dependence should be taken into account during the design and interpretation of results of in vitro T cell development assays and gene expression studies in vivo.

Research paper thumbnail of Adenosine deaminase deficiency increases thymic apoptosis and causes defective T cell receptor signaling

Journal of Clinical Investigation, 2001

Adenosine deaminase (ADA) deficiency in humans results in a severe combined immunodeficiency (SCI... more Adenosine deaminase (ADA) deficiency in humans results in a severe combined immunodeficiency (SCID). This immunodeficiency is associated with severe disturbances in purine metabolism that are thought to mediate lymphotoxicity. The recent generation of ADA-deficient (ADA(-/-)) mice has enabled the in vivo examination of mechanisms that may underlie the SCID resulting from ADA deficiency. We demonstrate severe depletion of T and B lymphocytes and defects in T and B cell development in ADA(-/-) mice. T cell apoptosis was abundant in thymi of ADA(-/-) mice, but no increase in apoptosis was detected in the spleen and lymph nodes of these animals, suggesting that the defect is specific to developing thymocytes. Studies of mature T cells recovered from spleens of ADA(-/-) mice revealed that ADA deficiency is accompanied by TCR activation defects of T cells in vivo. Furthermore, ex vivo experiments on ADA(-/-) T cells demonstrated that elevated adenosine is responsible for this abnormal TCR signaling. These findings suggest that the metabolic disturbances seen in ADA(-/-) mice affect various signaling pathways that regulate thymocyte survival and function. Experiments with thymocytes ex vivo confirmed that ADA deficiency reduces tyrosine phosphorylation of TCR-associated signaling molecules and blocks TCR-triggered calcium increases.

Research paper thumbnail of Highly Lytic CD8+, T-Cell Receptor Cytotoxic T Cells with Major Histocompatibility Complex (MHC) Class I Antigen-Directed Cytotoxicity in 2-Microglobulin, MHC Class I-Defiencient Mice

Research paper thumbnail of Generation of Antigen-Specific Murine CTL Under Weakly Immunogenic Conditions

Cytotoxic Cells: Recognition, Effector Function, Generation, and Methods, 1993

Research paper thumbnail of Multidrug-resistance phenotype of a subpopulation of T-lymphocytes without drug selection

Experimental Cell Research, 1989

Research paper thumbnail of A2A receptor dependent and A2A receptor independent effects of extracellular adenosine on murine thymocytes in conditions of adenosine deaminase deficiency

Blood, Jun 15, 2000

Adenosine deaminase (ADA) deficiency causes severe combined immunodeficiency (SCID) and is accomp... more Adenosine deaminase (ADA) deficiency causes severe combined immunodeficiency (SCID) and is accompanied by T-cell depletion and accumulation of both intracellular and extracellular adenosine (extAdo) and deoxyadenosine. To better understand the causes of T-cell depletion in vivo and to discriminate between extracellular and intracellular effects of exogenously added adenosine in vitro, we investigated mechanisms of 2 different effects of adenosine on murine thymocytes. These effects of adenosine include direct induction of apoptosis in about 6% to 15% thymocytes and inhibition of T-cell receptor TCR)-induced activation of the majority of thymocytes with inhibited ADA. A 2A adenosine receptors, but not A 2B , A 1 , or A 3 receptors, are shown to be mostly responsible for extAdo-triggered signaling (cyclic adenosine monophosphate [cAMP] accumulation) in murine thymocytes and this prompted studies of the effects of extAdo on thymocytes from A 2A R gene-deficient mice. It is found that direct apoptotic effects of extAdo on CD4 ؉ CD8 ؉ double positive (DP) thymocytes are completely accounted for by signaling through A 2A R, with no contribution of intracellular lymphotoxicity or of compensating A 2B Rs because only A 2A R ؉/؉, but not A 2A R ؊/؊ thymocytes were susceptible to apoptotic effects of ex-tAdo. Studies of the effects of cAMPraising agents support observations of extAdo/A 2A R/cAMP-triggered apoptosis in DP thymocytes. Unexpectedly, the extAdo strongly inhibited TCR-triggered activation of both A 2A R ؉/؉ and A 2A R ؊/؊ thymocytes in the presence of ADA inhibitors. This was confirmed with thymocytes from ADA gene-deficient mice, suggesting the existence of A 2A R-independent effects of extAdo on thymocytes. The presented data raises questions about the identity and functional role of A 2A R-expressing thymocytes in T-cell differentiation and of the role of TCRantagonizing effects of extAdo in conditions of ADA SCID. (Blood. 2000;95: 3859-3867)

Research paper thumbnail of Highly Lytic CD8^+, alphabeta T-Cell Receptor Cytotoxic T Cells with Major Histocompatibility Complex (MHC) Class I Antigen-Directed Cytotoxicity in beta_2-Microglobulin, MHC Class I-Defiencient Mice

Pnas, 1993

Targeted disruption of the beta 2-microglobulin (beta 2m) gene results in major histocompatibilit... more Targeted disruption of the beta 2-microglobulin (beta 2m) gene results in major histocompatibility complex (MHC) class I deficiency and virtual disappearance of functional CD8+ cytotoxic T lymphocytes (CTLs) in beta 2m-deficient (beta 2m-/-) mice. We asked whether the beta 2m-/- mice are able to reject tumor cells injected i.p. and what is the cellular composition of peritoneal exudate leukocytes (PELs) from such mice. We found that beta 2m-/- mice do reject MHC class I-bearing tumor cells injected i.p. Surprisingly, analysis of PEL CTLs obtained from i.p. tumor-injected beta 2m -/- mice revealed the presence of a large proportion of functional, tumor-destroying CD8+, CD4-, alpha beta T-cell receptor-positive, CD3+, Thy-1+, MHC class I-negative CTLs with strong MHC class I-directed cytotoxic activity. These results call for careful studies of local accumulation of CD8+ CTLs in beta 2m -/- mouse models and suggest that the dramatic decrease in MHC class I expression caused by beta 2m gene disruption does not prevent CD8+/CD4- cell selection and expansion.

Research paper thumbnail of Development and antigen specificity of CD8+ cytotoxic T lymphocytes in β2-microglobulin-negative, MHC class I-deficient mice in response to immunization with tumor cells

The Journal of Immunology, 1994

beta 2-Microglobulin knockout mice (beta 2-m-/-) with MHC class I expression deficiency are able ... more beta 2-Microglobulin knockout mice (beta 2-m-/-) with MHC class I expression deficiency are able to develop functional TCR(+)-alpha beta, CD8+ CTLs in response to tumor cell injection. The i.p. injection of beta 2-m-/- mice with tumor results in the massive accumulation of highly lytic CD8+ CTLs in the peritoneum and causes the local recruitment of CD8+ T cells into lymph nodes and spleens of immune animals. The accumulation of CD8+ CTLs in peritoneum is accompanied by the rejection of tumor cells and the survival of animals. The deficiency in MHC class I expression in beta 2-m/- mice is reflected in the delayed tumor rejection and CD8+ cell accumulation during the primary anti-tumor response in comparison with normal mice. The secondary response, however, is identical in normal and MHC class I-deficient mice. The rejection of tumor cells appears to be MHC class I directed because no rejection of tumors, no accumulation of CD8+ CTLs, and no survival of animals were observed when syngeneic tumor cells were used for injection with the notable exception of anti-minor Ag response. The Ag specificity of CD8+ CTLs in beta 2-m-/- mice is demonstrated using a panel of tumor target cells and class I transfectants. Although no substantial differences were found in the number and specificity of peritoneal CD8+ CTLs in beta 2-m-/- and normal mice using tumor rejection studies, the analysis of TCR-V beta phenotype using the panel of mAbs revealed the reduction in proportion of TCR-V beta 5 and TCR-V beta 6 used by CD8+ cell population from beta 2-m-/- mice. Development of lytic and H-2-directed CD8+ cells in regional lymph nodes was also observed after footpad immunization of beta 2-m-/- mice with TNP-labeled C57BL/6 splenocytes, suggesting anti-minor Ag reaction.

Research paper thumbnail of Critical Role of Hypoxia and A2A Adenosine Receptors in Liver Tissue-Protecting Physiological Anti-Inflammatory Pathway

Research paper thumbnail of Analysis of A2a receptor-deficient mice reveals no significant compensatory increases in the expression of A2b, Al, and A3 adenosine receptors in lymphoid organs

Biochemical Pharmacology, 2003

Although recent genetic and pharmacologic in vivo studies of acute inflammation models in mice de... more Although recent genetic and pharmacologic in vivo studies of acute inflammation models in mice demonstrated that the cyclic AMP-elevating A2a receptor plays a non-redundant role in protection from excessive acute inflammatory tissue damage and in the down-regulation of proinflammatory cytokine production, it remained to be established whether genetic deficiency of the A2a receptor is accompanied by a compensatory up-regulation of the cAMP-elevating A2b receptor and/or other adenosine receptors. Here, we show that most of the cAMP response to adenosine is abolished in lymphoid tissues of A2a receptor-deficient mice, although some response remains in splenocytes. No significant changes were observed in A2b, A1, and A3 mRNA levels in the thymus or lymph nodes of A2a receptor-deficient mice, but small increases in mRNA expression of these receptors were detected in the spleen. These data suggest that regulation of the expression of A2b, A1, and A3 receptors is not affected significantly by the absence of A2a receptors and may provide further explanation of earlier in vivo observations of increased tissue damage and of longer persistence of proinflammatory cytokines in animals with inactivated A2a receptors.

Research paper thumbnail of Role of A2a extracellular adenosine receptor-mediated signaling in adenosine-mediated inhibition of T-cell activation and expansion

Blood, Aug 15, 1997

(2) the A2a, but not the A1 or A3, receptors are the major absence of adenosine deaminase activit... more (2) the A2a, but not the A1 or A3, receptors are the major absence of adenosine deaminase activity (ADA) activity results in lymphocyte depletion and in severe combined im-expressed and functionally coupled adenosine receptors in mouse peripheral T and B lymphocytes, and the adenosine-munodeficiency (ADA SCID), which is currently explained by direct cell death-causing effects of intracellular products of induced cAMP accumulation in lymphocytes correlates with the expression of A2a receptors; (3) the specific agonist of adenosine metabolism. We explored the alternative mechanisms of peripheral T-cell depletion as due to inhibition of A2a receptor, CGS21680, induces increases in [cAMP]i in lymphocytes, whereas the specific antagonist of A2a recep-T-cell expansion by extracellular adenosine-mediated signaling through purinergic receptors. The strong inhibition of tor, CSC, inhibits the effects of Ado and CGS21680; and (4) the increases in [cAMP]i mimic the adenosine-induced inhi-the T-cell receptor (TCR)-triggered proliferation and of upregulation of interleukin-2 receptor a chain (CD25) mole-bition of TCR-triggered CD25 upregulation and splenocyte proliferation. These studies suggest the possible role of cules, but not the direct lymphotoxicity, were observed at low concentrations of extracellular adenosine. These effects adenosine receptors in the regulation of lymphocyte expansion and point to the downregulation of A2a purinergic re-of extracellular adenosine (Ado) are likely to be mediated by A2a receptor-mediated signaling rather than by intracellular ceptors on T cells as a potentially attractive pharmacologic target. toxicity of adenosine catabolites, because (1) poorly metabolized adenosine analogs cause the accumulation of cAMP ᭧ tively low concentrations of adenosine rather then the direct,

Research paper thumbnail of Studies of expression and possible functional role of purinergic receptors in cell-mediated immunity: Experimental approaches, controls, and caveats

Drug Develop Res, 1998

ABSTRACT

Research paper thumbnail of Purinoreceptors and T Cell Function: Regulation of Lymphocyte Activation by Extracellular ATP and Adenosine

Developments in Cardiovascular Medicine, 1998

Research paper thumbnail of Identification of Protein Kinases and Protein Phosphatases Involved in CTL Effector Functions. “ON” and “OFF” Signaling and Immunopharmacological Implications

Cytotoxic Cells: Recognition, Effector Function, Generation, and Methods, 1993

Research paper thumbnail of Critical role of hypoxia and A2A adenosine receptors in liver tissue-protecting physiological anti-inflammatory pathway

Molecular medicine (Cambridge, Mass.)

Whole body exposure of wild type control littermates and A2A adenosine receptor (A2AR) gene delet... more Whole body exposure of wild type control littermates and A2A adenosine receptor (A2AR) gene deleted mice to low oxygen containing inspired gas mixture allowed the investigation of the mechanism that controls inflammatory liver damage and protects the liver using a mouse model of T cell-mediated viral and autoimmune hepatitis. We tested the hypothesis that the inflammatory tissue damage-associated hypoxia and extracellular adenosine --> A2AR signaling plays an important role in the physiological anti-inflammatory mechanism that limits liver damage during fulminant hepatitis. After induction of T cell-mediated hepatitis, mice were kept in modular chambers either under normoxic (21% oxygen) or hypoxic (10% oxygen) conditions for 8 h. It was shown that the whole body exposure to hypoxic atmosphere caused tissue hypoxia in healthy animals as evidenced by a decrease in the arterial blood oxygen tension and increase of the plasma adenosine concentration (P < 0.05). This "hypoxic...

Research paper thumbnail of T cell-mediated immunity

Principles of Immunopharmacology, 2005

Research paper thumbnail of A2A adenosine receptor protects tumors from antitumor T cells

Proceedings of the National Academy of Sciences, 2006

The A2A adenosine receptor (A2AR) has been shown to be a critical and nonredundant negative regul... more The A2A adenosine receptor (A2AR) has been shown to be a critical and nonredundant negative regulator of immune cells in protecting normal tissues from inflammatory damage. We hypothesized that A2AR also protects cancerous tissues by inhibiting incoming antitumor T lymphocytes. Here we confirm this hypothesis by showing that genetic deletion of A2AR in the host resulted in rejection of established immunogenic tumors in Ϸ60% of A2ARdeficient mice with no rejection observed in control WT mice.

Research paper thumbnail of Transient up-regulation of P2Y2 nucleotide receptor mRNA expression is an immediate early gene response in activated thymocytes

Proceedings of the National Academy of Sciences, 1997

In studies designed to understand the roles of P2 nucleotide receptors in differentiation of T ly... more In studies designed to understand the roles of P2 nucleotide receptors in differentiation of T lymphocytes, we observed a transient and protein synthesis-independent enhancement of mRNA expression for the G protein-coupled P2Y 2 receptor in mouse thymocytes after the addition of steroid hormone or T cell receptor (TCR) crosslinking by anti-TCR mAb. Conversely, dexamethasone-induced increases in mRNA expression for the ligand-gated ion channel P2X 1 receptor was detected in rat, but not mouse, thymocytes, raising questions about the previously suggested role of P2X 1 receptors in thymocyte apoptosis. Flow cytometry analysis of thymocyte subsets excluded the possibility that the observed increases in P2Y 2 receptor mRNA expression were due to the enrichment of steroid-treated cells with an P2Y 2 mRNA-rich thymocyte subset. Triggering of TCR-mediated intracellular signaling pathways through crosslinking of TCR or by addition of phorbol ester and Ca 2؉ ionophore also resulted in the up-regulation of P2Y 2 , but not P2X 1 , receptor mRNA. It is proposed that the rapid increase of P2Y 2 receptor mRNA expression could be a common early event in responses of T cells to different activating stimuli. Taken together with the recently discovered ability of nucleotide receptor-initiated signaling to antagonize or enhance the effects of TCR crosslinking or steroids on thymocytes, the observed rapid up-regulation of P2Y 2 receptor mRNA expression may reflect an immediate early gene response where newly expressed cell surface nucleotide receptors provide regulatory feedback signaling from extracellular ATP in the T cell differentiation process.

Research paper thumbnail of A Serine/Threonine Phosphorylation Site in the Ectodomain of a T Cell Receptor β Chain is Required for Activation by Superantigen

Journal of Receptors and Signal Transduction, 2003

The presence of consensus phosphorylation sites in the ectodomains of cell surface proteins sugge... more The presence of consensus phosphorylation sites in the ectodomains of cell surface proteins suggests that such post-translational modification may be important in regulation of surface receptor activity. To date, the only cell surface receptor for which such ectodomain phosphorylation has been conclusively demonstrated is the clonally expressed T cell antigen receptor (TCR). Attempts to conclusively identify individual phosphorylated residues in TCR alpha and beta chains and determine their functional significance by biochemical approaches failed due to insufficient quantities of purified molecules. Here we present the results of an alternative approach where survey of phosphorylation sites in the TCR alpha and beta chains was accomplished using site-directed mutagenesis and retroviral vector expression, as well as in vitro phosphorylation of synthetic peptide substrates. All mutants studied directed the cell surface expression of normal amounts of TCR, and all transfectants could be stimulated to produce IL-2 in response to substrate-immobilized antibody to TCR. However, mutation of serine-88 in the protein kinase A phosphorylation site of the TCR beta chain resulted in a complete lack of response to the superantigen staphylococcal enterotoxin B (SEB). In addition, this mutation abolished TCR-associated tyrosine phosphorylation, consistent with the impairment of cell signaling. Reversion of the serine-88/alanine mutation with phosphorylatable threonine completely restored the SEB recognition by TCR. These results, interpreted in the context of the known three-dimensional structure of the complex of SEB and TCR, are consistent with the view that serine-88 is important for the contact of the TCR beta chain with SEB.

Research paper thumbnail of Cutting Edge: Physiologic Attenuation of Proinflammatory Transcription by the Gs Protein-Coupled A2A Adenosine Receptor In Vivo

The Journal of Immunology, 2004

Research paper thumbnail of Differential Effects of Physiologically Relevant Hypoxic Conditions on T Lymphocyte Development and Effector Functions

The Journal of Immunology, 2001

Direct measurements revealed low oxygen tensions (0.5-4.5% oxygen) in murine lymphoid organs in v... more Direct measurements revealed low oxygen tensions (0.5-4.5% oxygen) in murine lymphoid organs in vivo. To test whether adaptation to changes in oxygen tension may have an effect on lymphocyte functions, T cell differentiation and functions at varying oxygen tensions were studied. These studies show: 1) differentiated CTL deliver Fas ligand- and perforin-dependent lethal hit equally well at all redox conditions; 2) CTL development is delayed at 2.5% oxygen as compared with 20% oxygen. Remarkably, development of CTL at 2.5% oxygen is more sustained and the CTL much more lytic; and 3) hypoxic exposure and TCR-mediated activation are additive in enhancing levels of hypoxia response element-containing gene products in lymphocyte supernatants. In contrast, hypoxia inhibited the accumulation of nonhypoxia response element-containing gene products (e.g., IL-2 and IFN-gamma) in the same cultures. This suggests that T cell activation in hypoxic conditions in vivo may lead to different patterns of lymphokine secretion and accumulation of cytokines (e.g., vascular endothelial growth factor) affecting endothelial cells and vascular permeabilization. Thus, although higher numbers of cells survive and are activated during 20% oxygen incubation in vitro, the CTL which develop at 2.5% oxygen are more lytic with higher levels of activation markers. It is concluded that the ambient 20% oxygen tension (plus 2-ME) is remarkably well suited for immunologic specificity and cytotoxicity studies, but oxygen dependence should be taken into account during the design and interpretation of results of in vitro T cell development assays and gene expression studies in vivo.

Research paper thumbnail of Adenosine deaminase deficiency increases thymic apoptosis and causes defective T cell receptor signaling

Journal of Clinical Investigation, 2001

Adenosine deaminase (ADA) deficiency in humans results in a severe combined immunodeficiency (SCI... more Adenosine deaminase (ADA) deficiency in humans results in a severe combined immunodeficiency (SCID). This immunodeficiency is associated with severe disturbances in purine metabolism that are thought to mediate lymphotoxicity. The recent generation of ADA-deficient (ADA(-/-)) mice has enabled the in vivo examination of mechanisms that may underlie the SCID resulting from ADA deficiency. We demonstrate severe depletion of T and B lymphocytes and defects in T and B cell development in ADA(-/-) mice. T cell apoptosis was abundant in thymi of ADA(-/-) mice, but no increase in apoptosis was detected in the spleen and lymph nodes of these animals, suggesting that the defect is specific to developing thymocytes. Studies of mature T cells recovered from spleens of ADA(-/-) mice revealed that ADA deficiency is accompanied by TCR activation defects of T cells in vivo. Furthermore, ex vivo experiments on ADA(-/-) T cells demonstrated that elevated adenosine is responsible for this abnormal TCR signaling. These findings suggest that the metabolic disturbances seen in ADA(-/-) mice affect various signaling pathways that regulate thymocyte survival and function. Experiments with thymocytes ex vivo confirmed that ADA deficiency reduces tyrosine phosphorylation of TCR-associated signaling molecules and blocks TCR-triggered calcium increases.