Adenosine deaminase deficiency increases thymic apoptosis and causes defective T cell receptor signaling

Abstract

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.

Figure 1

Demonstration of extensive cell death in thymus but not in peripheral lymphoid organs of ADA^–/– mice. (a) Genetic and biochemical evidence of ADA deficiency in screening for ADA^+/+, ADA^+/–, and ADA^–/– mice. Littermates of ADA heterozygous mice were first analyzed by zymogram assay to identify ADA^–/– mice, and then tail DNA samples were analyzed by Southern blot as described in ref. . Arrowheads on the zymogram indicate position of ADA and hemoglobin (internal control). (b) Comparison of spleens and thymi from ADA^+/+ and ADA^–/– littermates. ×3. (c) Decreased cellularity of lymphoid organs of ADA^–/– mice. n, number of animals analyzed.

Figure 2

Increased apoptosis in the thymus but not in the lymph nodes or spleens of ADA^–/– mice. (a) Top: Side scatter versus forward scatter evaluation of proportion of dead cells in thymus of ADA^+/+ and ADA^–/– littermates. Numbers indicate percentage of live cells in the lymphoid organ. Bottom: Propidium iodide and Annexin V–aided evaluation of proportion of dead cells in thymus of ADA^+/+ and ADA^–/– littermates. Numbers indicate the percentage of live cells in the lower left gate estimated using both Annexin V and PI cell death flow cytometry assay as described in Methods. (b) Cytochemical demonstration of extensive clusters of apoptotic cells in thymus of ADA^–/– but not of ADA^+/+ littermates. Apoptotic cells are shown in red. The frozen tissue preparations and apoptosis detection were performed as described in Methods. (c) Flow cytometry demonstration of thymocyte distribution in thymi of ADA^–/– and ADA^+/+ littermates. (d) Flow cytometry demonstration of normal subset distributions in lymph nodes and spleens of ADA^–/– mice compared with organs of ADA^+/+ littermates. Numbers indicate the percentage of live cells in different subsets estimated as described in Methods.

Figure 3

Changes in phenotype of peripheral CD4⁺ and CD8⁺ T cells from spleens of ADA^–/– mice. Splenocytes of ADA^–/– and ADA^+/+ littermates were analyzed by three-color flow cytometry. Expression of different cell surface markers on CD4⁺ cells (a) and CD8⁺ cells (c) were analyzed by using CD4/CD8 gates as indicated by arrows in b. Numbers indicate proportions of cell from total cell population (b) or among gated cells (a and c).

Figure 4

Changes in phenotype of peripheral B cells from spleens of ADA^–/– mice. Splenocytes of ADA^–/– and ADA^+/+ littermates were analyzed by three-color flow cytometry as described in Methods and indicated by arrows. (a) Evaluation of B220⁺ B cells in ADA^–/– and ADA^+/+ littermates. Numbers represent percentage of B cells from total splenocytes. (b) Distribution of B cells among marginal, follicular, and newly formed zones of spleen in ADA^–/– versus ADA^+/+ mice. Numbers indicate percentage of cells in different zones. Cartoon illustrates location of different B cell subsets according to staining with mAb to CD21 and CD23.

Figure 5

Inhibited TCR-triggered activation of T cells in an ADA-deficient environment in vivo. ADA^+/+ and ADA^–/– littermates were injected intraperitoneally (i.p.) with PBS (control) or anti-CD3 mAb; 16 hours later, spleens were harvested and analyzed by flow cytometry for expression of T cell activation markers as described in Methods. Representative results of two (Exp. 1 and Exp. 2) of more than ten similar experiments using more than 20 pairs of littermates of ADA^–/– and ADA^+/+ mice are presented. (a) Comparison of TCR-triggered upregulation of CD25 and CD69 surface antigens on CD8⁺ T cells in an ADA^–/– or ADA^+/+ in vivo environment after injection of 5 μg of anti-CD3 mAb. (b) Comparison of TCR-triggered upregulation of CD25 and CD69 surface antigens on CD4⁺ T cells in an ADA-deficient or ADA-containing in vivo environment after injection of 5 μg of anti-CD3 mAb.

Figure 6

Effect of adenosine on TCR-triggered upregulation of CD25 and CD69 activation markers in vitro. Ex vivo spleen cells from ADA^–/– and ADA^+/+ littermates were incubated in 96-well plates with immobilized anti-CD3 mAb (5 μg/ml mAb) or with serum-free, ADA-free media alone (0 μg/ml mAb) in the presence or absence of adenosine (100 μM); 16 hours later, TCR-triggered upregulation of the T cell activation markers CD69 and CD25 was evaluated by flow cytometry.

Figure 7

Extracellular adenosine inhibits TCR-induced signaling in thymocytes. (a) Adenosine alone inhibits TCR-triggered apoptosis in ADA^–/– but not in normal ADA^+/+ thymocytes. (b) Demonstration of opposite effects of adenosine and 2′-deoxyadenosine on spontaneous and TCR-triggered apoptosis in ADA^–/– thymocytes. Thymocytes from wild-type or ADA^–/– mice were incubated for 16 hours in 96-well plates with apoptosis-inducing immobilized anti-CD3 mAb in the presence or absence of added adenosine (100 μM) or 2′-deoxyadenosine (100 μM). The effect of adenosine and 2′-deoxyadenosine on thymocyte survival (proportion of live cells) was evaluated using Annexin V assay as described in the Methods.

Figure 8

ADA deficiency is accompanied with defects in TCR signaling pathways in vivo and in vitro. (a) Partial phosphorylation of CD3 ζ chain is notably reduced in nonactivated ex vivo thymocytes from ADA^–/– mice, whereas ZAP-70 levels are shown to be the same in parallel samples of immunoprecipitates from ex vivo thymocytes harvested from ADA^–/– and ADA^+/+ littermates. (b) Intracellular Ca²⁺ mobilization upon ConA stimulation is inhibited by adenosine in normal thymocytes in the presence of the ADA inhibitor EHNA. Thymocytes from ADA^+/+ or ADA^–/– mice were used for isolation of ZAP-70 immunocomplexes followed by immunoblotting analysis of TCR ζ chain phosphorylation with an anti phosphotyrosine mAb as described in Methods. Anti ZAP-70 mAb’s were used in control immunoblotting. For measurements of calcium flux ADA^+/+ thymocytes were preloaded with indo-1 and analyzed on a FACSVantage flow cytometer as described in Methods. The percentages of cells that increase intracellular calcium after stimulation with Concanavalin A are shown on the graphs. Adenosine (100 μΜ) alone or in combination with EHNA (10 μM) was added a minute before the ConA stimulation. Arrow indicates time of injection of T cell–activating stimuli.