The extracellular versus intracellular mechanisms of inhibition of TCR-triggered activation in thymocytes by adenosine under conditions of inhibited adenosine deaminase

Sergey G. Apasov and Michail V. Sitkovsky

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


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The absence or low levels of adenosine deaminase (ADA) in humans result in severe combined immunodeficiency (SCID), which is characterized by hypoplastic thymus, T lymphocyte depletion and autoimmunity. Deficiency of ADA causes increased levels of both intracellular and extracellular adenosine, although only the intracellular lymphotoxicity of accumulated adenosine is considered in the pathogenesis of ADA SCID. It is shown that extracellular but not intracellular adenosine selectively inhibits TCR-triggered up-regulation of activation markers and apoptotic events in thymocytes under conditions of ADA deficiency. The effects of intracellular adenosine are dissociated from effects of extracellular adenosine in experiments using an adenosine transporter blocker. We found that prevention of toxicity of intracellular adenosine led to survival of TCR-cross-linked thymocytes in long-term (4 days) assays, but it was not sufficient for normal T cell differentiation under conditions of inhibited ADA. Surviving TCR-cross-linked thymocytes had a non-activated phenotype due to extracellular adenosine-mediated, TCR-antagonizing signaling. Taken together the data suggest that both intracellular toxicity and signaling by extracellular adenosine may contribute to pathogenesis of ADA SCID. Accordingly, extracellular adenosine may act on thymocytes, which survived intracellular toxicity of adenosine during ADA deficiency by counteracting TCR signaling. This, in turn, could lead to failure of positive and negative selection of thymocytes, and to additional elimination of thymocytes or autoimmunity of surviving T cells.

Keywords: adenosine deaminase, adenosine, apoptosis, G proteins, purinergic receptors, severe combined immunodeficiency disease, TCR, thymocytes


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The effects of adenosine on lymphocytes have been studied for more than two decades because of their role in the pathogenesis of diseases (14). More recently, adenosine has attracted attention due to its possible involvement in the regulation of normal T lymphocyte differentiation processes by virtue of its signaling through adenosine receptors (5,6). In addition, adenosine is being considered as an endogenous anti-inflammatory agent (7,8), while adenosine analogs are being developed for multipurpose use as pharmacologic agents (911).

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 26–28). 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.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Animals
DBA/2, chicken ovalbumin (OVA; peptide 323–339)-specific TCR transgenic D011.10 mice (32) were maintained in a pathogen-free environment at NIH animal care facilities. Mice were 6–10 weeks old and two to three animals were used in each experiment.

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 5x10–5 M ß2-mercaptoethanol. B cell lymphoma A20 cells (H-2d) were maintained in RPMI complete media and used as antigen-presenting cells (APC) for OVA–TCR transgenic thymocytes.

mAb
R-phycoerythrin (PE)-conjugated hamster anti-mouse {alpha}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 (0.5–1x106 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 323–339-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 16–18 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, 0.6–1x106 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 V–FITC 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).


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Extracellular adenosine inhibits the antigenic peptide–TCR-induced death of thymocytes
We tested whether extracellular adenosine may play a role in the thymocyte depletion observed during ADA SCID by incubating ex vivo thymocytes from normal or TCR-transgenic mice with extracellular adenosine in the presence or absence of ADA inhibitors. Antigenic peptide–TCR-triggered apoptosis assays were used in the experiments described in Fig. 1Go.




View larger version (48K):
[in this window]
[in a new window]
 
Fig. 1. Effect of extracellular adenosine on TCR-triggered apoptosis in thymocytes. Extracellular adenosine antagonizes the TCR-induced apoptosis of thymocytes in the absence but not in the presence of normal levels of ADA activity. TCR-transgenic thymocytes were incubated ex vivo in the absence (control) or presence of antigen-presenting A20 (I-Ad ) cells and various concentrations of synthetic antigenic peptide. Adenosine was added at the beginning of culture in the presence or absence of ADA inhibitors. (A) Thymocyte apoptosis is blocked by extracellular adenosine if ADA activity is partially inhibited by a low concentration (2 µM) of the ADA inhibitor coformycin. Concentrations of exogenous adenosine are indicated on the graphs. Thymocytes were incubated for 16 h with or without coformycin and the percent of surviving cells was estimated using flow cytometry analysis by forward/side scatter profiles and PI staining. Coformycin (cof) alone had little or no effect on thymocyte survival compared with media alone. (B) Inhibition of TCR-triggered apoptosis of thymocytes by a low concentration (0.1 mM) of extracellular adenosine in the presence of 10 µM of ADA inhibitor (coformycin or EHNA). Thymocytes were incubated 12 h and analyzed by the annexin V-based flow cytometry apoptosis assay. Surviving cells were defined as PI negative and annexin V-staining negative. Effects of extracellular adenosine on survival of TCR-transgenic thymocytes were examined in the presence of different concentrations of specific antigenic peptide as indicated. The addition of inosine in the parallel control had no effect.

 
It has been described (32) and is confirmed here (Fig. 1Go) that incubation of thymocytes from TCR-transgenic mice with various concentrations of antigenic cOVA (323–329) peptide and A20 B cell lymphoma (I-Ad) as APC results in peptide concentration-dependent death of thymocytes. Only ~40% of thymocytes survived exposure to 1 µM of antigenic peptide during a 16 h in vitro assay (Fig. 1AGo). To mimic conditions of ADA deficiency, we used two different and well-characterized inhibitors of ADA, coformycin (36,37) and EHNA (38). In control experiments, we confirmed the ability of these drugs to efficiently inhibit the activity of ADA in mouse thymocytes and determined the dose–response dependence of effects of these inhibitors (data not shown). In the experiment described in Fig. 1Go(A), 2 µM of coformycin was used to model conditions of partial ADA inactivation, since this concentration was lower than that routinely employed for complete inhibition of ADA. It is shown that coformycin alone did not have any effect on TCR-triggered apoptosis (Fig. 1AGo). Similarly, the addition of extracellular adenosine alone at 0.2, 1.0 and 5.0 mM did not have an effect on peptide-triggered thymocyte death.

Remarkably, the simultaneous addition of both coformycin and extracellular adenosine resulted in increased survival of peptide–APC-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 peptide–TCR-induced death, instead of being lymphotoxic.

Protection of thymocytes from antigenic peptide–TCR-induced death at lower concentrations of extracellular adenosine was observed when complete inhibition of ADA was accomplished using higher concentrations of ADA inhibitors (Fig. 1BGo). Complete protection from TCR-triggered cell death was achieved by as little as 100 µM adenosine in this experiment. While ~50–60% 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. 2Go). It is shown in Fig. 2Go 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. 1Go that extracellular adenosine—under 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).



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 2. The combined addition of adenosine and of ADA inhibitor EHNA prevents selective elimination of transgenic TCR-expressing CD4+CD8+ thymocytes by antigenic peptide. Thymocytes were incubated with 10 nM of OVA peptide as described in Fig. 1Go(A) and stained with FITC-conjugated anti-transgenic TCR-specific mAb (KJ-1-26) and with PE-conjugated anti-CD8 mAb (data not shown). Histograms indicate intensity of TCR expression versus relative cell number among surviving thymocytes.

 
Of importance, very low or no TCR-antagonizing effects of extracellular adenosine were observed if no ADA inhibitor was added. This points to both the high efficiency of normal levels of ADA in degrading extracellular adenosine and the need for sustained increased local levels of extracellular adenosine to accomplish the TCR-antagonistic effects observed during ADA deficiency. Indeed, no survival of thymocytes above control (APC alone, no antigenic peptide levels) was observed, even at 1 mM extracellular adenosine, if added alone. To the contrary, the addition of adenosine to untreated (no APC–peptide) thymocytes in the absence of ADA inhibitor resulted in a slow-developing and protein synthesis-dependent apoptosis of predominantly CD4+CD8+ thymocytes in parallel samples (data not shown) (32).

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. 1BGo). 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-TCR–CD3 complex mAb (Fig. 3AGo). 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.



View larger version (38K):
[in this window]
[in a new window]
 
Fig. 3. Extracellular adenosine rescues thymocytes from anti-CD3 mAb- and PMA-induced apoptosis with no protection from anti-Thy-1 or anti-Fas mAb-induced apoptosis. Thymocytes were incubated in vitro with 10 µg/ml immobilized anti-CD3 mAb (A) or with 10 nM PMA (B) or in plates covered with various concentrations of anti-Thy-1 mAb or anti-FAS mAb (C) and anti-CD3 (D) for 16 h. Addition of adenosine transporter inhibitor NBTI (10 µM) had no effect on the ability of extracellular adenosine to block anti-CD3-induced apoptosis in thymocytes (D). Adenosine (100 µM) was added at the beginning of culture in the absence or presence of ADA inhibitor EHNA (10 µM). The numbers of apoptotic and surviving cells were estimated by flow cytometry after thymocytes were stained with PI and annexin V–FITC.

 
This protective effect was not reversed by addition of adenosine transporter inhibitor NBTI and it was observed at various concentrations of anti-CD3 stimuli (Figs 3D and 5DGoGo). Data in Fig. 3Go support the extracellular mechanism of adenosine action. The anti-CD3 mAb were not as efficient as antigenic peptide–APC (Fig. 1Go) or phorbol myristate acetate (PMA) (Fig. 3BGo) in triggering apoptosis in thymocytes, but strong protective effects of extracellular adenosine were observed even with PMA-treated thymocytes (Fig. 3BGo). Only 25–30% of thymocytes survived exposure to 10 nM PMA in control incubation or in the presence of adenosine, inosine, ADA inhibitor EHNA or inosine and EHNA; however, ~60% of thymocytes survived the apoptotic effects of PMA when both extracellular adenosine and EHNA were included.




View larger version (94K):
[in this window]
[in a new window]
 
Fig. 5. Inhibition of adenosine transporter by NBTI improves survival of thymocytes under conditions of ADA deficiency. Thymocytes from DBA/2 mice were incubated ex vivo in plates covered with anti-CD3 mAb, which were immobilized on plastic at a concentration of 20 µg/ml. After 16 (A), 40 (B) or 96 (C) h of incubation, cells were analyzed by flow cytometry using annexin V, anti-CD69 and PI staining of the cells as described in Methods. Percentages of live thymocytes (annexin V and PI negative) are indicated. Extracellularly added adenosine (0.1 mM), in combination with coformycin (10 µM) and NBTI (10 µM), protects cells from anti-CD3 mAb-induced apoptosis. These reagents alone or in any other combination were not effective (data not shown). (D) Thymocytes were incubated with anti-CD3 mAb immobilized on plastic at concentrations of 1.25, 5 and 20 µg/ml for 16 or 40 h, and the percentage of surviving cells was calculated.

 
In contrast, the `rescuing' effects of extracellular adenosine were not observed with other apoptotic stimuli (Fig. 3CGo). Even in the presence of ADA inhibitor, extracellular adenosine did not antagonize and even enhanced the Fas-mediated (39) death pathway in thymocytes. Similarly, no protective effects were detected against anti-CD45 mAb-induced apoptosis (40) (data not shown) and anti-Thy-1 mAb-induced apoptosis (41) (Fig. 3CGo). Indeed, it was shown that Thy-1-mediated apoptosis of thymocytes is different from the TCR-triggered pathway of `activation-driven cell death' (41). In agreement with these data, extracellular adenosine and ADA inhibitors were not able to protect thymocytes from `spontaneous' death when cells were cultured in the absence of APC and OVA peptide (Fig. 1AGo). The inability of extracellular adenosine to antagonize Fas- and Thy-1-mediated apoptosis is consistent with its selective effects in protection from TCR-mediated signaling. In fact, under these conditions, anti-FAS-induced apoptosis was more effective.

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. 1Go). 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. 4Go, we found that extracellular adenosine inhibits mitogen- (Fig. 4A and BGo) and peptide–TCR-induced (Fig. 4CGo) 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, Gotop panel) or EHNA (Fig. 4AGo, 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. 4BGo) 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.




View larger version (43K):
[in this window]
[in a new window]
 
Fig. 4. Extracellular adenosine inhibits TCR- or mitogen-triggered up-regulation of CD69 activation markers on thymocytes in vitro. Thymocytes from DBA/2 mice were incubated for 4 and 22 h with or without Con A (2.5 µg/ml), extracellular adenosine (0.1 mM) and ADA inhibitors (10 µM), and then analyzed by flow cytometry after staining with FITC-conjugated anti-CD69 mAb. (A) Up-regulation of CD69 is inhibited by extracellular adenosine under conditions of inhibited ADA activity. Both ADA inhibitors coformycin and EHNA were used. (B) Expression of CD69 in mitogen-activated thymocytes after 22 h incubation with or without ADA inhibitor. Percentages of CD69-expressing cells after Con A stimulation and in the presence of adenosine, inosine and ADA inhibitors are indicated on the histograms. (C) TCR-triggered up-regulation of CD69 is inhibited by extracellular adenosine under conditions of inhibited ADA activity. Thymocytes from OVA–TCR-transgenic mice were incubated for 4 h with 1 µM of antigenic peptide, APC, extracellular adenosine and EHNA as described in Fig. 1Go, and then analyzed by flow cytometry. A20 cells were preincubated with antigenic peptide for 30 min and were used as APC. The cAMP analog, db-cAMP (100 µM), and a selective agonist of A2a adenosine receptor, CGS 21680 (10 µM), were included in parallel assays instead of extracellular adenosine and EHNA.

 
Extracellular adenosine was also efficient in inhibiting peptide–TCR-triggered CD69 up-regulation on OVA–TCR-transgenic thymocytes (28–13%) (Fig. 4CGo).

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 1–3GoGoGo). 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 peptide–APC–TCR-triggered CD69 up-regulation on TCR-transgenic thymocytes (Fig. 4CGo). Similarly, a selective agonist of A2a adenosine receptors (CGS21680) was able to inhibit TCR-induced activation of thymocytes.

The ability of cAMP (Fig. 4CGo) to mimic effects of extracellular adenosine (Figs 1–4GoGoGoGo) 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 6GoGo). 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).



View larger version (34K):
[in this window]
[in a new window]
 
Fig. 6. Inhibition of adenosine transporter by NBTI does not prevent the TCR-antagonizing effects of extracellular adenosine under conditions of ADA deficiency and surviving thymocytes are not activated. Cells were cultured and stained as described in Fig. 5Go. Histograms show early activation marker CD69 on live (annexin V/PI negative) thymocytes after 16 h, 40 h and 4 days of incubation of thymocytes in wells without (control) or with immobilized anti-CD3 mAb (20 µg/ml). The expression of CD69 was compared among thymocytes that were activated by anti-CD3 mAb in the presence or absence of extracellular adenosine (100 µM), coformycin (10 µM), and NBTI (10 µM). Effects of adenosine + coformycin + NBTI are compared with the effects of adenosine + coformycin and with the effects of each of these compounds added alone.

 
It is shown that extracellular adenosine antagonizes TCR-triggered activation even when transport of extracellular adenosine is blocked by nucleotide transporter blockers, thus supporting the extracellular signaling model of effects of extracellular adenosine under conditions of ADA deficiency. Most likely, NBTI actually prolongs the effects of extracellular adenosine by preventing transport of extracellular adenosine into thymocytes and thereby enhancing the TCR-inhibiting effect of adenosine.

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. 5BGo) and even 4 days (Fig. 5CGo), but not after the first 16 h (Fig. 5AGo). 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. 5BGo).

The TCR-antagonizing effects of extracellular adenosine were observed at all tested concentrations of anti-CD3 mAb up to 20 µg/ml (Fig. 5DGo). 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. 6Go). In these experiments, thymocytes were triple stained with annexin V–FITC, PI and CD69 mAb–PE. 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. 5DGo).

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.


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Understanding the mechanisms of ADA SCID is of substantial interest, and is dependent on clarification of the effects of increased concentrations of adenosine on T cell differentiation and effector functions.

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. 3CGo). 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 6GoGo, 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. 5CGo), while surviving TCR-cross-linked thymocytes had a non-activated phenotype (Fig. 6CGo) 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 (6,47–49 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 16–96 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 ADA–CD26 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 TCR–antigen 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.


    Acknowledgments
 
We thank Drs Elizabeth Payer-Suri and Robert Ortmann (NIH, Bethesda) for screening transgenic mice and for the gift of FITC-labeled KJ-1-26 mAb respectively; Brenda Rae Marshall for editorial help; and Shirley Starnes for help in preparation of the manuscript.


    Abbreviations
 
A2a receptorsubtype of extracellular adenosine purinergic receptors
ADAadenosine deaminase
APCantigen-presenting cell
Con Aconcanavalin A
db-cAMPN6,2-O-dibutyryladenosine 3':5' cyclic monophosphate
EHNAerythro-9(2-hydroxy-3-nonyl)adenine hydrochloride
NBTIS-(4-nitrobenzyl)-6-thioinosine
OVAovalbumin
PEphycoerythrin
PIpropidium iodide
PMAphorbol myristate acetate
SCIDsevere combined immunodeficiency

    Notes
 
Transmitting editor: C. Terhorst

Received 10 August 1998, accepted 20 October 1998.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Giblett, E. R., Anderson, J. E., Cohen, F., Pollara, B. and Meuwissen, H. 1972. Adenosine-deaminase deficiency in two patients with severely impaired cellular immunity. Lancetii :1067.
  2. Kellems, R. E., Yeung, C.-Y. and Ingolia, D. E. 1985. Adenosine deaminase deficiency and severe combined immunodeficiencies. Trends Genet. 1:278.[ISI]
  3. Hershfield, M. S. and Mitchell, B. S. 1995. Immunodeficiency diseases caused by adenosine deaminase deficiency and purine nucleoside phosphorylase deficiency. In Scriver, C. R., Beaudet, A. L., Sly, W. S. and Valle D., eds, The Metabolic and Molecular Basis of Inherited Disease, p. 1725. McGraw-Hill, New York.
  4. Hirschhorn, R. 1995. Adenosine deaminase deficiency: molecular basis and recent developments. Clin. Immunol. Immunopathol. 76:S219.[ISI][Medline]
  5. Apasov, S., Koshiba, M., Redegeld, F. and Sitkovsky, M. 1995. Role of extracellular ATP and P1 and P2 classes of purinergic receptors in T-cell development and cytotoxic T lymphocyte effector functions. Immunol. Rev. 146:5.[ISI][Medline]
  6. Huang, S., Koshiba, M., Apasov, S. and Sitkovsky, M. 1997. Role of A2a adenosine receptor mediated signaling in inhibition of T-cells activation and expansion. Blood 90:1600.[Abstract/Free Full Text]
  7. Cronstein, B. N., Naime, D. and Firestein, G. 1995. The antiinflammatory effects of adenosine kinase inhibitor are mediated by adenosine. Arthritis Rheum. 38:1040.[ISI][Medline]
  8. Cronstein, B. N. 1995. A novel approach to the development of anti-inflammatory agents: adenosine release at the inflamed sites. J. Invest. Med. 43:50.[ISI][Medline]
  9. Jacobson, K. A., van Galen, P. J. M. and Williams, M. 1992. Perspective, adenosine receptors: pharmacology, structure activity relationships and therapeutic potential. J. Med. Chem. 35:407.[ISI][Medline]
  10. Olah, M. E. and Stiles, G. L. 1995. Adenosine receptor subtypes: characterization and therapeutic regulation. Annu. Rev. Pharmacol. Toxicol. 35:581.[ISI][Medline]
  11. Mizumura, T., Auchampach, J. A., Linden, J., Bruns, R. F. and Gross, G. J. 1996. PD 81,723, an allosteric enhancer of the A1 adenosine receptor, lowers the threshold for ischemic preconditioning in dogs. Circ. Res. 79:415.[Abstract/Free Full Text]
  12. Kohn, D. B. and Weinberg, K. I. 1996. Adenosine deaminase deficiency. In The Genes of Primary Immunodeficiency—Windows on the Immune System and Prospects for Gene Therapy, p. 7. NIAID, NIH, Bethesda, MD.
  13. Carson, D. A., Kaye, J. and Wasson, D. B. 1981. The potential importance of soluble deoxynucleotidase activity in mediating deoxyadenosine toxicity in human lymphoblasts. J. Immunol. 126:348.[Abstract/Free Full Text]
  14. Ullman, B., Levinson, B. B., Hershfield, M. S. and Martin, D W. 1981. A biochemical genetic study of the role of specific nucleoside kinases in deoxyadenosine phosphorylation by cultured human cells. J. Biol. Chem. 256:848.[Free Full Text]
  15. Doherty, P., Pan, S., Milloy, J., Thompson, E., Thorner, P., Barankiewicz, J., Roifman, C. and Cohen, A. 1991. Adenosine deaminase and thymocyte maturation. Scand. J. Immunol. 33:405.[ISI][Medline]
  16. Blackburn, M. R., Datta, S. K., Wakamiya, M., Vartabedian, B. S. and Kellems, R. E. 1996. Metabolic and immunologic consequences of limited adenosine deaminase expression in mice. J. Biol. Chem. 271:15203.[Abstract/Free Full Text]
  17. Liu, X., Kim, C. N., Yang, J., Jemmerson, R. and Wang, X. 1996. Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c. Cell 86:147.[ISI][Medline]
  18. Schwartz, A. L., Stern, R. C. and Polmar, S. H. 1978. Demonstration of an adenosine receptor on human lymphocytes in vitro and its possible role in the adenosine deaminase-deficient form of severe combined immunodeficiency. Clin. Immunol. Immunopathol. 9:499.[ISI][Medline]
  19. Jacobson, K. A., von Lubitz, D. K., Daly, J. W. and Fredholm, B. B. 1996. Adenosine receptor ligands: differences with acute versus chronic treatment. Trends Pharmacol. Sci. 17:108.[ISI][Medline]
  20. Kizaki, H., Suzuki, K., Tadakuma, T. and Ishimura, Y. 1990. Adenosine receptor-mediated accumulation of cyclic AMP-induced T lymphocyte death through internucleosomal DNA cleavage. J. Biol. Chem. 265:5280.[Abstract/Free Full Text]
  21. van der Ploeg, I., Ahlberg, S., Parkinson, F. E., Olsson, R. A. and Fredholm, B. B. 1996. Functional characterization of adenosine A2 receptors in Jurkat cells and PC12 cells using adenosine receptor agonists. Naunyn Schmiedebergs Arch. Pharmacol. 353:250.[ISI][Medline]
  22. Zenzer, T. V. 1975. Formation of adenosine 3',5'-monophosphate from adenosine in mouse thymocytes. Biochim. Biophys. Acta 404:202.[ISI][Medline]
  23. McConkey, D. J., Nicotera, P. and Orrenius, S. 1994. Signalling and chromatin fragmentation in thymocyte apoptosis. Immunol. Rev. 142:343.[ISI][Medline]
  24. Szondy, Z. 1994. Adenosine stimulates DNA fragmentation in human thymocytes by Ca2+-mediated mechanisms. Biochem. J. 304:877.[ISI][Medline]
  25. Jondal, M., Okret, S. and McConkey, D. 1993. Killing of immature CD4+CD8+ thymocytes in vivo by anti-CD3 or 5'-(N-ethyl)-carboxamide adenosine is blocked by glucocorticoid receptor antagonist RU-486. Eur. J. Immunol. 23:1246.[ISI][Medline]
  26. Kammer, G. M. 1988. The adenylate cyclase-cAMP-protein kinase A pathway and regulation of the immune response. Immunol. Today 9:222.[ISI][Medline]
  27. Takayama, H., Trenn, G. and Sitkovsky, M. V. 1988. Locus of inhibitory action of cAMP-dependent protein kinase in the antigen-receptor triggered cytotoxic T-lymphocyte activation pathway. J. Biol. Chem. 263:2330.[Abstract/Free Full Text]
  28. McConkey, D. J., Orrenius, S. and Jondal, M. 1990. Agents that elevate cAMP stimulate DNA fragmentation in thymocytes. J. Immunol. 145:1227.[Abstract/Free Full Text]
  29. Sugiyama, H., Chen, P., Hunter, M., Taffs, R. and Sitkovsky, M. 1992. The dual role of the cAMP-dependent protein kinase C{alpha} subunit in T-cell receptor-triggered T-lymphocyte effector functions. J. Biol. Chem. 267:25256.[Abstract/Free Full Text]
  30. Sugiyama, H., Chen, P., Hunter, M. and Sitkovsky, M. 1997. Perturbation of the expression of the catalytic subunit C{alpha} of PKA inhibits TCR-triggered secretion of IL-2 by T-helper cells. J. Immunol. 158:171.[Abstract]
  31. Lalli, E., Sassone-Corsi, P. and Ceredig, R. 1996. Block of T lymphocyte differentiation by activation of the cAMP-dependent signal transduction pathway. EMBO J. 15:528.[Abstract]
  32. Iwabuchi, K., Nakayama, K., McCoy, R. L., Wang, F., Nishimura, T., Habu, S., Murphy, K. M. and Loh, D. Y. 1992. Cellular and peptide requirements for in vitro clonal deletion of immature thymocytes. Proc. Natl Acad. Sci. USA 89:9000.[Abstract]
  33. Apasov, S. G., Koshiba, M., Chused, T. M. and Sitkovsky, M. V. 1997. Effects of extracellular ATP and adenosine on different thymocyte subsets. Possible role of ATP-gated channels and G-protein coupled receptors. J. Immunol. 158:5095.[Abstract]
  34. Darzynkiewicz, Z., Bruno, S., Del Bino, G., Gorczyca, W., Hotz, M. A., Lassota, P. and Traganos, F. 1992. Features of apoptotic cells measured by flow cytometry. Cytometry 13:795.[ISI][Medline]
  35. Martin, S. J., Reutelingsperger, C. P. M., McGahon, A. J., Rader, J. A., van Shie, R. C. A. A., LaFace, D. M. and Green, D. R. 1995. Early redistribution of plasma membrane phosphatidilserine is a general feature of apoptosis regardless of initiating stimulus: inhibition by overexpression of bcl-2 and abl. J. Exp. Med. 182:1545.[Abstract]
  36. Hall, J. G., Gyure, L., Peppard, J. and Orlans, E. 1979. Levels of adenosine deaminase in some experimental animal tumours and the possible therapeutic effect of the ADA inhibitor 2-deoxy-coformycin. Br. J. Cancer 40:750.[ISI][Medline]
  37. Hershfield, M. S. 1984. Conversion of a stem cell leukemia from a T-lymphoid to a myeloid phenotype induced by the adenosine deaminase inhibitor 2'-deoxycoformycin. Proc. Natl Acad. Sci. USA 81:253.[Abstract]
  38. Muraoka, T., Katsuramaki, T., Shiraishi, H. and Yokoyama, M. M. 1990. Automated enzymatic measurement of adenosine deaminase isoenzyme activities in serum. Anal. Biochem. 187:268.[ISI][Medline]
  39. Nagata, S. 1997. Apoptosis by death factor. Cell 88:355.[ISI][Medline]
  40. Klaus, S. J., Sidorenko, S. P. and Clark, E. A. 1996. CD45 ligation induces programmed cell death in T and B lymphocytes. J. Immunol. 156:2743.[Abstract]
  41. Hueber, A., Raposo, G., Pierres, M. and He, H.-T. 1994. Thy-1 triggers mouse thymocyte apoptosis through a bcl-2-resistant mechanism. J. Exp. Med. 179:785.[Abstract]
  42. Testi, R., D'Ambrosio, D., De Maria, R. and Santoni, A. 1994. The CD69 receptor: a multipurpose cell-surface trigger for hematopoietic cells. Immunol. Today 15:479.[ISI][Medline]
  43. Hugo, P., Kappler, J. W. and Marrack, P. C. 1993. Positive selection of TcR {alpha}ß thymocytes: is cortical thymic epithelium an obligatory participant in the presentation of major histocompatibility complex protein? Immunol. Rev. 135:133.[ISI][Medline]
  44. Ashton-Rickardt, P. G., Bandeira, A., Delaney, J. R., Van Kaer, L., Pircher, H. P., Zinkernagel, R. M. and Tonegawa, S. 1994. Evidence for a differential avidity model of T cell selection in the thymus. Cell 76:651.[ISI][Medline]
  45. Surch, C. D. and Sprent, J. 1994. T-cell apoptosis detected in situ during positive and negative selection in thymus. Nature 372:100.[ISI][Medline]
  46. Yamazaki, T., Arase, H., Ono, S., Ohno, H., Watanabe, H. and Saito, T. 1997. A shift from negative to positive selection of autoreactive T cells by the reduced level of TCR signal in TCR-transgenic CD3 zeta-deficient mice. J. Immunol. 158:1634.[Abstract]
  47. Koshiba, M., Kojima, H., Huang, S., Apasov, S. and Sitkovsky, M. V. 1997. Memory of extracellular adenosine/A2a purinergic receptor-mediated signalling in murine T cells. J. Biol. Chem. 272:25881.[Abstract/Free Full Text]
  48. Dinjens, W. N., van Doorn, R., van Laarhoven, J. P., Roos, D., Zeijlemaker, W. P. and de Bruijn, C. H. 1986. Adenosine receptors on human T lymphocytes and human thymocytes. Adv. Exp. Med. Biol. 195B:1.
  49. Marone, G., Plaut, M. and Lichtenstein, L. M. 1978. Characterization of a specific adenosine receptor on human lymphocytes. J. Immunol. 121:2153.[Abstract]
  50. Resta, R., Hooker, S. W., Laurent, A. B., Rahman, J. S. M., Franklin, M., Knudsen, T. B., Nadon, N. L. and Thompson, L. F. 1997. Insights into thymic purine metabolism and adenosine deaminase deficiency revealed by transgenic mice overexpressing ecto-5'-nucleotidase (CD73). J. Clin. Invest. 15:676.
  51. Kameoka, J., Tanaka, T., Nojima, Y., Schlossman, S. F. and Morimoto, C. 1993. Direct association of adenosine deaminase with a T cell activation antigen, CD26. Science 261:466.[ISI][Medline]
  52. Shortman, K. and Scollay, R. 1994. Death in the thymus. Nature 372:44.[ISI][Medline]
  53. Vacchio, M. S., Papadopoulos, V. and Ashwell, J. D. 1994. Steroid production in the thymus: implications for thymocyte selection. J. Exp. Med. 179:1835.[Abstract]
  54. Robey, E. and Fowlkes, B. J. 1994. Selective events in T cell development. Annu. Rev. Immunol. 12:675.[ISI][Medline]