Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, 10 Center Drive-MSC 1892, National Institutes of Health, Bethesda, MD 20892-1892 USA
Correspondence to: M. V. Sitkovsky
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Abstract |
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Keywords: adenosine deaminase, adenosine, apoptosis, G proteins, purinergic receptors, severe combined immunodeficiency disease, TCR, thymocytes
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Introduction |
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Accumulation of extracellular and intracellular adenosine in the absence of adenosine deaminase (ADA) activity is lymphotoxic and causes severe combined immunodeficiency (ADA SCID). ADA deficiency results in hypoplastic thymus, and a decrease in the number of peripheral lymphocytes in lymph nodes and spleen (14). In addition, ADA SCID patients suffer from an autoimmune disorder (12) that was interpreted as an imbalance between immune effector and immune regulatory cells.
While the deficiency of ADA causes increased levels of both intracellular and extracellular adenosine, only the intracellular lymphotoxicity of accumulated adenosine is considered to contribute to the pathogenesis of ADA SCID. Indeed, the most widely accepted mechanism of ADA SCID implicates formation of intracellular deoxyadenosine, deoxyATP and/or S-adenosyl homocysteine as well as pyrimidine starvation (3,4,1317) as the direct cause of intracellular lymphotoxicity.
The alternative `signaling' mechanism of thymocyte depletion during ADA SCID needs to be investigated, because extracellular adenosine can initiate transmembrane signaling through purinergic receptors on thymocytes and on peripheral T cells (5,6,1820). Extracellular adenosine receptors (P1 class) are expressed in many tissues, and include Gs or Gi protein-coupled A1, A2a, A2b and A3 receptors (reviewed in 19,21). It has been shown that extracellular adenosine induced increases in cAMP (22) or intracellular [Ca2+] and apoptosis in mouse or human thymocytes (20,2325), but patterns of expression and identification of individual classes of adenosine receptors in different subsets of lymphocytes have not been described. The Gi protein-coupled A1 receptors and Gs protein-coupled A2a and A2b receptors are known to modulate levels of cAMP (19,21). cAMP was long considered an important modulator of lymphocyte functions (reviewed in 2628). It was shown that the cAMP-dependent protein kinase-mediated biochemical pathway strongly affects the TCR-triggered processes in both peripheral T cells (2730) and thymocytes (31).
The clarification of the effects of extracellular adenosine under conditions of ADA deficiency is important for devising strategies for treatment of this diseases and for rational prediction of side effects of adenosine-based pharmacologic agents.
Taken together, these considerations prompted us to evaluate and to discriminate between the effects of extracellular versus intracellular adenosine on TCR-activated thymocytes in short- and long-term in vitro assays under conditions of inhibited ADA. This revealed the TCR-antagonizing effects of extracellular adenosine on thymocytes which may contribute to the mechanisms of T cell depletion and autoimmunity in ADA SCID.
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Methods |
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Cells and medium
Thymocytes were isolated from adult thymus ex vivo and incubated in RPMI 1640 (Biofluids, Rockville, MD) supplemented with 5% dialyzed FCS (heat inactivated), and 100 U/ml penicillin, 100 µg/ml streptomycin, 1 mM sodium pyruvate, 1 mM HEPES, non-essential amino acids and 5x105 M ß2-mercaptoethanol. B cell lymphoma A20 cells (H-2d) were maintained in RPMI complete media and used as antigen-presenting cells (APC) for OVATCR transgenic thymocytes.
mAb
R-phycoerythrin (PE)-conjugated hamster anti-mouse ß-TCR (H57-597) and rat anti-mouse CD4 (RM-4-5), and FITC, and PE-conjugated rat anti-mouse CD8 mAb (clone 53-67) were purchased from PharMingen (San Diego, CA). Rat anti-mouse CD4 (H129.19) mAb conjugated with Red-613 fluorochrome were purchased from Gibco/BRL/Life Technologies (Gaithersburg, MD).
Reagents
Adenosine was prepared freshly as 20 mM stock solution. Adenosine and adenosine analogs NECA, CGS216680, CSC and 2-chloro-adenosine, as well as ADA inhibitor erythro-9(2-hydroxy-3-nonyl)adenine hydrochloride (EHNA) and nucleotide transporter inhibitors S-(4-nitrobenzil)-6-thioinosine (NBTI) and dipyridamole, were purchased from RBI (Natick, MA). N6,2-O-dibutyryladenosine-3',5'-cyclic monophosphate (db-cAMP) was purchased from Sigma (St Louis, MO). Coformycin was purchased from Calbiochem (San Diego, CA). Selective A2a receptor antagonist ZM241385 was a kind gift from Drs Joel Linden (University of Virginia, Charlottesville, VA) and Kenneth Jacobson (NIH, Bethesda). It was also purchased from Tocris Cookson (Ballwin, MO).
Analysis of thymocytes
The use of TCR-transgenic thymocytes provided internal controls for the specificity of effects of extracellular adenosine on TCR-triggered versus untriggered thymocytes. A single-cell suspension of murine thymocytes was isolated by standard procedures and cultured in 96-well plates (0.51x106 cells/well) as described (33). To analyze the effects of adenosine in the experimental model of negative thymic selection in vitro (32), we used thymocytes from transgenic mice that express OVA peptide 323339-specific TCR DO11.10. Apoptosis of TCR-transgenic thymocytes was induced as described (32) using antigen-presenting A20 cells that expressed I-Ad MHC class II molecules and presented specific antigenic peptide. After incubation for 1618 h or as indicated, cells were harvested and analyzed by flow cytometry.
Flow cytometric quantitation of live, apoptotic and dead cells was done according to a modified flow cytometry procedure (34) as described (33). The effects of adenosine on thymocytes were studied after incubating thymocytes ex vivo in short-term culture. The analysis of surviving, dead and apoptotic cells was based on gating of cells by size (side and forward scatter), plasma membrane integrity [propidium iodide (PI) staining] and redistribution of plasma membrane phosphatidylserine (annexin V binding). The annexin V binding assay was done as described (35). Briefly, 0.61x106 cells from a 96-well plate were resuspended in 100 µl of buffer containing 10 mM HEPES, pH 7.3, 150 mM NaCl, 5 mM KCl, 1 mM MgCl2 and 1.8 mM CaCl2, and incubated with 0.3 µg/ml of FITC-conjugated annexin V and 5 µg/ml PI for 15 min. After incubation, samples were diluted 4 times with buffer containing 1.8 mM CaCl2 and analyzed by FACScan. Annexin VFITC was purchased from Trevingen (Gaithersburg, MD) and Biowhittaker (Walkersville, MD).
Statistical analysis of triplicate sample measurements was performed using the StatView statistic program (Abacus Concepts, Berkeley, CA). SD of triplicate measurements within the same experiment usually were <1%.
Flow cytometry data acquisition and analysis were done on a FACScan using FACScan research software and CellQuest programs (Becton Dickinson, San Jose, CA).
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Results |
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Remarkably, the simultaneous addition of both coformycin and extracellular adenosine resulted in increased survival of peptideAPC-exposed thymocytes. About 30% of thymocytes were `rescued' with 0.2 mM of adenosine in the presence of 2 µM of coformycin during incubation with the antigenic peptide and APC, while the addition of 1 mM of adenosine resulted in virtually complete protection of thymocytes, even at concentrations of antigenic peptide that caused maximal (~70%) thymocyte death. Even at very high concentrations such as 5 mM, extracellular adenosine rescued thymocytes from peptideTCR-induced death, instead of being lymphotoxic.
Protection of thymocytes from antigenic peptideTCR-induced death at lower concentrations of extracellular adenosine was observed when complete inhibition of ADA was accomplished using higher concentrations of ADA inhibitors (Fig. 1B). Complete protection from TCR-triggered cell death was achieved by as little as 100 µM adenosine in this experiment. While ~5060% of thymocytes were counted as apoptotic after incubation with 1 µM of antigenic peptide, the presence of both 100 µM adenosine and 10 µM coformycin or EHNA resulted in complete survival of thymocytes.
The protection of TCR-transgenic immature thymocytes from peptide- and APC-induced apoptosis in the presence of both coformycin (10 µM) and adenosine (100 µM) is also demonstrated using a clonotypic marker for transgenic TCR (anti-KJ-1-26 mAb) (Fig. 2). It is shown in Fig. 2
that only in the presence of both extracellular adenosine and ADA inhibitor was the number of transgenic TCR-expressing cells not decreased. This confirms observations in Fig. 1
that extracellular adenosineunder conditions of ADA deficiency rescues thymocytes that would be otherwise deleted by antigenic peptide and APC. The flow cytometry analysis of these cells by double and triple staining with anti-CD4 and anti-CD8 mAb confirmed that the same immature transgenic TCR-expressing CD4+CD8+ TCR KJ-1-26+ thymocytes that were predominantly targeted for apoptosis by antigenic peptide (32) were protected from apoptosis by extracellular adenosine (data not shown).
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In agreement with the extracellular mechanism of action of extracellular adenosine, the product of adenosine degradation by ADA, inosine, was not active in parallel control samples either alone or together with ADA inhibitor (Fig. 1B). Similarly, no changes in protective effects of extracellular adenosine or enhancement of thymocyte survival were detected when translocation of extracellular adenosine in the cytoplasm was blocked by inhibitors of adenosine transporters (dipyridamole or NBTI; data not shown).
The ability of extracellular adenosine to counteract TCR effects could be demonstrated not only with the antigenic peptide but also with anti-TCRCD3 complex mAb (Fig. 3A). Incubation with anti-CD3 mAb is shown to trigger apoptosis in thymocytes, which was almost completely antagonized by the combined addition of extracellular adenosine and EHNA in a parallel experiment. Again, both extracellular adenosine and ADA inhibitor, but neither alone, were able to block effects of TCR cross-linking on thymocytes.
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Extracellular adenosine inhibits TCR-triggered up-regulation of early thymocyte activation markers
The dramatic `rescuing' effects of extracellular adenosine described above suggested that extracellular adenosine acts as a signaling molecule rather than as an intracellular lymphotoxic intermediate. Indeed, even as much as 5 mM extracellular adenosine did not have strong lymphotoxic effects on thymocytes (Fig. 1). We interpret our data as suggesting that apoptosis-antagonizing `rescuing' effects of extracellular adenosine are mediated by its interference with TCR-triggered activation of thymocytes. Therefore, it was expected that extracellular adenosine would inhibit TCR-triggered expression of activation markers on thymocytes. In experiments described in Fig. 4
, we found that extracellular adenosine inhibits mitogen- (Fig. 4A and B
) and peptideTCR-induced (Fig. 4C
) up-regulation of CD69 surface antigen, which is used in thymocyte studies as an early T cell activation marker (42). Antagonistic effects of combined addition of extracellular adenosine and of ADA inhibitors coformycin (Fig. 4A,
top panel) or EHNA (Fig. 4A
, bottom panel) on up-regulation of thymocyte activation marker CD69 were detected as early as 4 h after the start of incubation and were very pronounced after prolonged incubation. It is shown that concanavalin A (Con A; 2.5 µg/ml) causes increased expression of activation marker CD69 (from 6 to up to 63% in Fig. 4B
) on the cell surface of thymocytes after 22 h of culture. The addition of adenosine (0.1 mM) in the presence of ADA inhibitor (10 µM) at the beginning of culture results in a strong reduction of CD69+ thymocytes to ~20%, and most of those thymocytes have very low levels of CD69 expression. Neither extracellular adenosine alone nor ADA inhibitor EHNA alone has such inhibitory effects. Control samples with inosine, the product of adenosine deamination, had no effect, either.
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The described TCR-antagonistic effects of extracellular adenosine and the increased survival of thymocytes could not be explained by the action of lymphotoxic intracellular catabolites of adenosine. Indeed, we demonstrate here that even high concentrations of extracellular adenosine, instead of being lymphotoxic, actually improved thymocyte survival or even completely protected thymocytes from TCR-triggered apoptosis (Figs 13). Taken together, these data suggest that the effects of extracellular adenosine are most likely due to general inhibition of TCR-triggered signaling pathways. The most straightforward assumption was that extracellular adenosine antagonizes TCR signaling by activation of the cAMP-dependent pathway. As noted above, there is a large body of evidence that cAMP increases do, in fact, inhibit TCR signaling (2631). Indeed, the cAMP analog db-cAMP was mimicking inhibitory effects of extracellular adenosine on peptideAPCTCR-triggered CD69 up-regulation on TCR-transgenic thymocytes (Fig. 4C
). Similarly, a selective agonist of A2a adenosine receptors (CGS21680) was able to inhibit TCR-induced activation of thymocytes.
The ability of cAMP (Fig. 4C) to mimic effects of extracellular adenosine (Figs 14
) was in agreement with the possibility that extracellular adenosine antagonizes TCR signaling in thymocytes by transmembrane signaling through the P1 class of adenosine receptors.
Inhibition of adenosine transport by nucleotide transporter blockers does not affect TCR-antagonizing effects of extracellular adenosine but protects thymocytes from toxic effects of intracellular adenosine
In the next series of experiments, we used blockers of adenosine transporters to test the possibility that the extracellularly added adenosine is not acting on thymocytes by signaling through adenosine receptors but is rather transported into the cytoplasm, where intracellular toxic metabolites of adenosine inhibit the TCR-induced effects in thymocytes. This was tested by incubating thymocytes with activating anti-CD3 mAb, adenosine and ADA inhibitor in the presence or absence of adenosine transport inhibitor NBTI in a time course experiment (Figs 5 and 6). NBTI was used to discriminate between the extracellular and intracellular action of extracellular adenosine on thymocytes. In parallel control experiments, we confirmed using a fluorescent adenosine analog that NBTI does prevent transport of adenosine into thymocytes (data not shown).
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It is shown that the presence of NBTI protects thymocytes from cell death under conditions of increased adenosine and of ADA deficiency by blocking the adenosine transporter-mediated accumulation of toxic intracellular adenosine. The most profound effects of NBTI could be seen after 40 h (Fig. 5B) and even 4 days (Fig. 5C
), but not after the first 16 h (Fig. 5A
). After, for example, 40 h in vitro, only 12% of anti-CD3 mAb-treated thymocytes were alive, while 60% of anti-CD3 mAb-treated thymocytes (the same as in media alone) survived TCR incubation in the presence of extracellular adenosine and NBTI (Fig. 5B
).
The TCR-antagonizing effects of extracellular adenosine were observed at all tested concentrations of anti-CD3 mAb up to 20 µg/ml (Fig. 5D). The simultaneous addition of adenosine transporter inhibitor NBTI with extracellular adenosine resulted in complete blocking of thymocyte apoptosis.
To test whether surviving thymocytes had an activated phenotype, the effects of extracellular adenosine on activation of TCR-triggered thymocytes in the presence of NBTI were tested in a thymocyte activation assay using activation marker CD69 (Fig. 6). In these experiments, thymocytes were triple stained with annexin VFITC, PI and CD69 mAbPE. This allowed us to analyze CD69 expression by excluding not only dead but also early apoptotic cells.
It is shown in time course studies that anti-CD3 mAb do induce a progressively higher proportion of live thymocytes to express increased levels of CD69 after 16, 40 and 96 h of incubation in vitro (Fig. 5D).
In agreement with the extracellular mechanism of action, addition of extracellular adenosine completely blocks CD69 up-regulation. These effects of extracellular adenosine are not changed by inclusion of NBTI in the incubation media. In control experiments, adenosine, ADA inhibitor or NBTI alone had no effect.
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Discussion |
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While the intracellular lymphotoxicity of accumulated adenosine was considered in explanations of pathogenesis of ADA SCID, the alternative (or additional) mechanism of extracellular adenosine-mediated signaling through purinergic receptors was not investigated.
In this report, we describe experiments designed to discriminate between extracellular and intracellular mechanisms of adenosine action. It is shown that extracellular but not intracellular adenosine has properties that may be the cause of some of the features observed in ADA SCID. Our observations of TCR-antagonistic effects of extracellular adenosine on thymocytes offer an dditional explanation of T cell depletion and of autoimmunity in ADA SCID patients by suggesting dual, intracellular versus extracellular mechanisms of action at increased concentrations of adenosine.
The main observation is that extracellular adenosine selectively inhibits TCR-triggered signaling and blocks the up-regulation of early activation markers in thymocytes, such as CD69 of TCR-transgenic and normal thymocytes in short-term cultures ex vivo. Moreover, both TCR- and PMA-triggered apoptosis of thymocytes were abrogated or dramatically inhibited by extracellular adenosine in the presence of ADA inhibitors. Of importance, the effects of extracellular adenosine were selective against TCR stimuli but not against other tested cell surface-initiated apoptotic stimuli, including mAb to Fas, CD45 and Thy-1 surface proteins (Fig. 3C). In fact, anti-Fas mAb-mediated apoptosis of thymocytes was enhanced by the presence of extracellular adenosine and ADA inhibitors.
The following `signaling' model of ADA SCID is consistent with the data presented here. (i) As the result of ADA deficiency, thymocytes are exposed to increased concentrations of both intracellular and extracellular adenosine, which exert different effects on thymocytes. (ii) Accumulation of intracellular adenosine is toxic and, depending on its levels in individual cells, eventually is lethal to many but not all thymocytes. However, thymocytes which survived intracellular toxicity are exposed to effects of extracellular adenosine. (iii) Accumulated extracellular adenosine acts on thymocytes by signaling through adenosine receptors and counteracting TCR signaling that is all-important for thymocyte selection.
Accordingly, under conditions of low or no ADA activity, the accumulated extracellular adenosine may prevent the TCR-driven positive selection of thymocytes, which then will die from `neglect' (4345). Subsequently, in the absence of positive selection, no mature thymocytes will develop and the number of peripheral T cells will be greatly reduced. On the other hand, some thymocytes with very high avidity of TCR, which are normally eliminated by negative selection, may be spared and develop as autoreactive cells. This view is supported by the recent demonstration of a shift from negative to positive selection of autoreactive T cells by reduced levels of TCR signaling in the model of TCR-transgenic mice (46).
This model is supported by described here dissociation of toxic effects of intracellular adenosine from TCR-inhibiting effects of extracellular adenosine, which was accomplished using the inhibitor of adenosine transporter, NBTI. As is shown in Figs 5 and 6, the majority of TCR-activated thymocytes were rescued from death during long-term (4 day) incubation in vitro under conditions of inhibited ADA by blocking the adenosine transporter-mediated accumulation of toxic intracellular adenosine (Fig. 5C
), while surviving TCR-cross-linked thymocytes had a non-activated phenotype (Fig. 6C
) because of extracellular adenosine-mediated, TCR-antagonizing signaling.
It remains to be determined which of the observed overall changes in thymocytes and T cells in ADA SCID are due to extracellular versus intracellular mechanisms of adenosine action. Any proposed mechanism must explain both the depletion of a large proportion of T cells and thymocytes, and the inability of surviving T cells to mount a normal immune response. Indeed, if the remaining thymocytes and T cells in ADA SCID patients were functionally normal, they could be expanded during a specific antigen-driven response. Our experiments with inhibited ADA suggest that the depletion of a large proportion of thymocytes is due in large part to the intracellular toxicity of adenosine and its catabolites, while the TCR-inhibiting effects of extracellular adenosine may contribute to immunodeficiency by additional decreases in thymocyte survival and by changes in the qualitative composition in the TCR repertoire of those thymocytes that survive. This abnormal thymocyte selection may result both in the disappearance of some TCR, and in the survival of autoimmune thymocytes and T cells that otherwise would be negatively selected.
Thus, the `signaling' model of ADA SCID may explain the T cell subset-specific changes and autoimmunity observed in ADA SCID patients that otherwise are not compatible with an intracellular, `metabolic' mechanism of adenosine action. Indeed, clinical observations of autoimmunity in ADA SCID patients have been explained (12) as an imbalance between immune effector and immune regulatory cells, implying differential susceptibility of effector and regulatory T cell subsets. Our observations suggest that effects of extracellular adenosine could lead to survival and maturation of harmful T cells and autoimmunity in some patients with ADA SCID. The subset-specific mode of adenosine action was also suggested by observations of predominantly immature (CD3lowCD4CD8) circulating T cells in ADA SCID patients due to the block in thymocyte differentiation (12).
The mechanism of extracellular adenosine signaling on thymocytes is yet to be determined. A2a adenosine receptors are among the candidates to mediate effects of extracellular adenosine. Our experiments did demonstrate the A2a receptor mediated accumulation of cAMP in thymocytes (data not shown) and the cAMP pathway is well known to inhibit TCR-driven processes in T cells (27,29,30). The time course of adenosine-induced cAMP accumulation is such that much more profound effects of adenosine could be expected in the absence of ADA activity. Indeed, the mandatory requirement to have both ADA inhibitor (to prevent degradation of extracellular adenosine) and extracellular adenosine to see physiologic effects of adenosine in thymocytes is consistent with results of studies of cAMP accumulation in which we determined (6,4749 and data not shown) that in the absence of ADA inhibitor, the single addition of adenosine is sufficient to induce significant increases in cAMP levels in thymocytes only for about 30 min to 1 h. After that, levels of cAMP decrease. Thus, the presence of ADA inhibitor seems to be required to ensure the prolonged presence of extracellular adenosine is required to observe the sustained signaling and physiologic effects in 1696 h assays. By extension, only the inhibition of ADA (or its deficiency) in vivo may lead to the sustained levels of extracellular adenosine necessary to cause sufficient signaling and effects in long-term thymocyte response.
Future experiments with adenosine receptor-deficient animals may shed light on the relative contributions of individual classes of adenosine receptors, including A2 and A3 receptors in effects of extracellular adenosine.
Recent studies of ADA-deficient mice (16) and transgenic mice overexpressing 5' nucleotidase (50) provide the opportunity to model the ADA SCID phenomena and to evaluate the role of ADA in T cell development. The availability of such animals and samples from patients with ADA SCID may provide an appropriate system to test whether there are changes in the TCR repertoire in conditions of ADA deficiency.
The data reported here also support the possibility of adenosine receptor involvement in the regulation of normal T cell differentiation in the thymic environment by antagonizing signals from TCR in thymocytes and the importance of ADA in the control of adenosine-triggered signaling in thymocytes and T cells. While the expression of ADA dramatically decreases as thymocytes mature from prothymocytes and cortical thymocytes to medullary thymocytes into peripheral T cells (15), the exact thymocyte subset distribution of ADA, as well as the role of ADACD26 association in T cells (51), remains to be determined. Adenosine receptor-expressing thymocyte subsets with low ADA activity are expected to be especially sensitive to the effects of extracellular adenosine-mediated signaling.
Considerations of extracellular adenosine functions attract attention to the largely unexplored possibility of a general mechanism of regulation of lymphocyte differentiation and effector functions by small, extracellular, non-immune molecules which could signal through G protein-coupled receptors or membrane channels on thymocytes (33,41) to counteract or to enhance the effects of TCRantigen interactions. Signaling by non-immune factors could be either direct or in combination with TCR-mediated effects. Similarly to steroids (53), the purinergic receptor-mediated action of extracellular adenosine could contribute to the processes of positive and negative selection of thymocytes that result in the death of the majority of thymocytes (45) and promote differentiation of immature thymocytes into mature T cells (54).
Thus, in addition to their role in ADA SCID, the physiologically abundant molecules of extracellular nucleotides could serve as non-immune factors in the `normal' thymic environment. Indeed, both TCR-dependent and -independent mechanisms appear to be involved in the regulation of extensive cell death in the thymus and in the selection of thymocytes (52). Furthermore, ADA was recently shown to exist in direct association with the ectodomain of T cell activation antigen CD26 (51).
Finally, the model of ADA SCID described here provides one more incentive for the development of adenosine-based drugs and suggests the extracellular receptor as a potential pharmacologic target for immunomodulation.
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Acknowledgments |
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Abbreviations |
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A2a receptor | subtype of extracellular adenosine purinergic receptors |
ADA | adenosine deaminase |
APC | antigen-presenting cell |
Con A | concanavalin A |
db-cAMP | N6,2-O-dibutyryladenosine 3':5' cyclic monophosphate |
EHNA | erythro-9(2-hydroxy-3-nonyl)adenine hydrochloride |
NBTI | S-(4-nitrobenzyl)-6-thioinosine |
OVA | ovalbumin |
PE | phycoerythrin |
PI | propidium iodide |
PMA | phorbol myristate acetate |
SCID | severe combined immunodeficiency |
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Notes |
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Received 10 August 1998, accepted 20 October 1998.
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References |
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