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International Immunology, Vol. 15, No. 9, pp. 1105-1116, September 2003
© 2003 Japanese Society for Immunology

Death of T cell precursors in the human thymus: a role for CD38

Claudya Tenca1, Andrea Merlo1, Daniela Zarcone1, Daniele Saverino1, Silvia Bruno1, Amleto De Santanna2, Dunia Ramarli5, Marina Fabbi4, Carlo Pesce3, Silvia Deaglio6, Ermanno Ciccone1, Fabio Malavasi6 and Carlo E. Grossi1

Sections of 1 Human Anatomy and 2 Histology, Department of Experimental Medicine, and 3 Department of Biophysics and Dental Sciences, University of Genova, 16132 Genova, Italy 4 Cancer Research Institute, Genova, Italy 5 Institute of Immunology and Infectious Diseases, University of Verona, Verona, Italy 6 Laboratory of Immunogenetics, University of Torino, Torino, Italy

The first two authors contributed equally to this work
Correspondence to: C. E. Grossi, Department of Experimental Medicine, Institute of Human Anatomy, University of Genova, Via De Toni 14, 16132 Genova, Italy. E-mail: anatuman{at}unige.it
Transmitting editor: S. Izui


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Thymic T cell maturation depends on interactions between thymocytes and cells of epithelial and hematopoietic lineages that control a selective process whereby developing T cells with inappropriate or self-reactive receptors die. Molecules involved in this process are the TCR expressed on thymocytes together with the CD3 complex and MHC–peptide on accessory cells. However, other molecules may favor or prevent death of thymocytes, thus playing a role in selection. CD38 is expressed by the majority of human thymocytes, mainly at the double-positive (DP) stage. In contrast, CD38 is not found on subcapsular double-negative (DN) thymocytes and on a proportion of medullary single-positive (SP) thymocytes. CD38 enhances death of thymocytes when it is cross-linked by goat anti-mouse (GAM) antiserum or by one of its ligands, CD31, expressed by thymic epithelial cells or transfected into murine fibroblasts (L cells). As most thymocytes are at an intermediate (DP) stage of development, it is likely that these cells are most vulnerable to death mediated via MHC–peptide–TCR interactions that is increased by CD38 cross-linking. DN and SP thymocytes are refractory to CD38-induced apoptosis. Accessory molecules, e.g. CD38, are expressed during thymic cell maturation and their presence is relevant for the survival or death of DP T cells in the course of selection. Based on our data, CD38 enhances thymocyte death by interacting with CD31 expressed by accessory cells. In addition, CD28 expression on developing thymocytes also appears to play a role for their selection and it synergizes with CD38 to induce apoptosis of DP thymocytes.

Keywords: CD31, CD38, apoptosis, thymocytes


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
T cell precursors mature in the embryonic and post-natal thymus where they undergo genetic and phenotypic changes that yield a repertoire of mature T lymphocytes equipped with TCR and accessory molecules that activate or inhibit their functions (1–4). Relevant to T cell development within the thymus is a series of cell–cell interactions between maturing thymocytes and a variety of accessory cells that comprise endoderm/ectoderm-derived epithelial cells and cells of hematopoietic lineage, such as interdigitating cells and macrophages (5–9). The thymic epithelium comprises of at least three distinct subsets, i.e. subcapsular ‘nurse’ cells, cortical dendritic and medullary cells (10–12). These cells exert different functions by sustaining T lymphoblast proliferation, and the processes of positive and negative selection that reshape the repertoire by eliminating cells equipped with inefficient or self-reactive receptors respectively (13–16). Thus, interaction of thymocytes with non-lymphoid cells dictates a series of maturational events that ensue T cell survival or death (4,17–19).

T cell development involves two steps of selection. The rescue of double-positive (DP) thymocytes from programmed cell death allows their maturation into CD4 or CD8 single-positive (SP) cells, and the process is known as positive selection. This event is mediated by thymic epithelial cells (TEC). Positive selection ensures that all mature T cells express functional receptors capable of responding to peptides presented by self-MHC molecules on antigen-presenting cells. Cells programmed for passive apoptosis (i.e. by neglect) are rescued by this type of selection.

Thymocytes also undergo negative selection that eliminates self-reactive cells. This death process depends largely on interdigitating cells and macrophages, and is mediated by an activation-induced (active) cell death.

Molecular interactions that regulate T cell selection depend mainly on the expression of specific TCR with the CD3-associated complex, and on their affinity for peptides presented by accessory cells in the context of class I and II MHC molecules. Among them, TECs are the largest component and their interaction with developing thymocytes is central to the selective process (20–22).

CD38 is a surface molecule expressed by human immature and activated T and B lymphocytes, monocytes, and NK cells. It is a type II membrane receptor that exerts ADP ribosyl cyclase activity. This molecule is involved in the transduction of activation and proliferation signals, and participates in the adhesion of lymphocytes to endothelium via its ligand, CD31 (27–29).

Other molecules expressed by thymocytes and their ligands on epithelial cells could, however, exert a role for the end result of thymic selection. These molecular interactions occur during extensive cell–cell adhesive contacts in the course of T cell ontogeny (7,23–26). Among them, CD28 could also play a role in the selective process.

In this study, we investigated the expression and functional role of CD38 found on the majority of human thymocytes, mainly at the DP stage (i.e. CD4+ and CD8+, CD1a+). In contrast, CD38 was undetectable in double-negative (DN) (i.e. subcapsular) thymocytes and in a proportion of medullary (mature) thymocytes. One of the CD38 ligands, CD31, is expressed by thymocytes and is also found in the thymic epithelium (30–34). On functional grounds, cross-linking of CD38 by goat anti-mouse (GAM) antiserum and interaction with its ligand (CD31) enhances apoptotic death of thymocytes at the DP stage. At variance, CD31 cross-linking does not seem to affect significantly the rate of thymocyte death.

Experiments in this study indicate that, apart from MHC–TCR interactions, CD38 sustains the massive T cell death that occurs in the thymus. This is possibly due to its association with CD3, as shown previously for phytohemagglutinin (PHA)-activated thymocytes (35) and confirmed in this study using freshly isolated resting cells. Our results indicate that CD38 may be involved in thymic selection. Although controversial, it appears that engagement of TCR or CD3{epsilon} alone is unable to induce thymocyte apoptosis (14). Therefore, a second signal is necessary and it appears that several molecules (e.g. CD28 or CD38) may serve this role. We also show that CD28 synergizes with CD38 to induce apoptosis of DP thymocytes.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Cells
Thymocytes were obtained by mechanical disruption of thymus fragments from 14 pediatric patients undergoing corrective cardiac surgery. Viable cells (>95%) were obtained from thymocyte suspensions by Ficoll-Hypaque density gradient centrifugation, washed and used immediately. Cells were maintained in RPMI 1640 with 10% FCS, 5 mM L-glutamine and 50 IU/ml penicillin–streptomycin.

DN thymocytes were isolated from thymocyte suspensions using anti-human CD4- and CD8-coated microbeads (Miltenyi Biotec, Auburn, CA) followed by magnet separation. As DP (CD4+ and CD8+, and CD1a+) cells comprise the majority of thymocytes (>=95%, see Fig. 1), whole cell suspensions were considered as representative of this intermediate stage of T cell development.



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  		Fig. 1. Surface phenotype of thymocytes at different maturation stages. DN thymocytes were obtained from freshly prepared thymus cell suspensions depleted of CD4+ and CD8+ cells. Of note is the dim expression of CD3, CD38 and CD31 on a small proportion of cells. CD4, CD8 and CD1a are undetectable. Fresh thymocyte suspensions are largely enriched for DP cells as shown by the presence of CD1a, and co-expression of CD4 and CD8. CD38 and CD31, as well as CD3, are up-regulated. SP cells were obtained by immunomagnetic sorting (see Methods). Both CD4+ and CD8+ SP thymocytes express CD3 brightly, and CD1a is absent. A small contaminant fraction (6%) is present in both SP subsets. The phenotype of PBL is also shown for comparison.

 
SP thymocytes were obtained from thymocyte suspensions using anti-human CD1a-coated microbeads (Miltenyi Biotec, Auburn, CA) followed by magnetic separation. CD1a– thymocytes were further separated using anti-human CD4- and CD8-coated microbeads and magnet. The resulting SP subsets were checked by flow cytometry using anti-CD1a/CD4/CD8 mAb. Contaminant cells were ~6% (see Fig. 1). Furthermore, in order to generate mature SP cells, thymocytes were pulsed with PHA (5 µg/ml) and cultured with human recombinant IL-2 (at 50 U/ml final concentration) up to 60 days (36).

Peripheral blood lymphocytes (PBL) were obtained from healthy donors by Ficoll-Hypaque density gradient centrifugation.

TEC cultures were derived and expanded as described previously (25,26).

Murine fibroblasts, L cells, transfected with the human CD31 gene or mock-transfected, were cultured in DMEM with 10% FCS, 5mM L-glutamine and 50 IU/ml penicillin–streptomycin (30).

Antibodies
The following antibodies were used for immunofluorescence assays, thymocyte cultures and immunohistochemistry: anti-CD38 (clone IB4, IgG2a; IB6, IgG2b; SUN-4B7, IgG1; OKT10, IgG1); anti-CD158b1 (clone A3, IgG1); anti-LAIR-1 (clone DX26, IgG1, kindly provided by Dr Joe Philips, DNAX, Stanford, CA); anti-CD31 (clone Moon-1, IgG1); anti-CD28 (clone CB28, IgG1); anti-CD1a (clone SK9, IgG2a; Becton Dickinson, San Jose, CA); anti-CD2 (clone RPA-2.10, IgG1; Becton Dickinson); anti-CD3{epsilon} (UCH-T1, IgG1); anti-CD4 (OKT4, IgG2a); anti-CD8 (OKT8, IgG2b; ATCC, Rockville, MD); anti-MHC class I (W6/32, IgG2a, ATCC); anti-cytokeratin (clone CAM5.2, IgG2A; Becton Dickinson).

Immunophenotypic analyses
The surface phenotype of thymocytes and TEC was evaluated by flow cytometry (FACSCalibur; Becton Dickinson). The secondary reagents were phycoerythrin- or FITC-labeled GAM antisera (Southern Biotechnology Associates, Birmingham, AL).

For cytoplasmic immunostaining, TEC were fixed with 4% paraformaldehyde and permeated using 0.1% saponin, before labeling with anti-CD38, anti-CD31 and anti-cytokeratin mAb. Negative controls were provided by cells incubated with the secondary reagent alone.

Co-capping experiments
Thymocytes (0.5 x 106) were incubated with anti-CD38 or anti-CD3{epsilon} mAb for 30 min on ice, washed and allowed to react with GAM isotype conjugated with CY3 (Jackson ImmunoResearch, West Baltimore Pike, PA) for 30 min on ice. Samples were washed and incubated at 37°C for 2 h to induce capping. Cells were then treated with the alternative mAb (to CD3 or CD38) for 20 min on ice. Finally, thymocytes were incubated with GAM isotype conjugated with Alexa Fluor 488 (Molecular Probes, Eugene, OR) to detect co-localization. Cells were fixed with 1% paraformaldehyde, cytocentrifuged and analyzed with an epifluorescence inverted microscope (Olympus, Hamburg, Germany). The same experiments were performed using anti-MHC class I as an irrelevant mAb and an anti-CD2 mAb as isotype-matched control. Images were acquired using a chilled Hamatsu CCD black and white camera, and processed with IP-LAB and Adobe Photoshop software.

Evaluation of apoptosis
Apoptosis assays were performed using fresh thymocyte suspensions cultured in 48-well plates at 2–3 x 106 cells/ml in the presence of anti-CD3{epsilon}, anti-CD38, anti-CD28 or -CD31 mAb, with or without GAM antiserum. Antibodies were added in the culture at 1 µg/ml and GAM antiserum was used at 3 µg/ml. Apoptosis was evaluated after 24, 48 and 72 h of culture by flow cytometric analysis after double staining with FITC–Annexin V and propidium iodide, according to the manufacturer’s instructions (Bender MedSystems, Vienna, Austria). In this two-color analysis, Annexin V binding to the cell membrane indicates early apoptotic events, whereas propidium iodide staining shows cell permeability to the dye and, thus, progression from apoptosis to necrosis. Cells at these stages of death are detected in the lower right and in the upper right quadrants of the FACS profiles, respectively. Four anti-CD38 mAb were used and they were able to induce thymocyte apoptosis at the same rate. Apoptosis induced by anti-CD28 mAb was also evaluated.

To assess apoptosis of fresh SP thymocytes, purified CD4+ and CD8 + thymocytes obtained by two-step immunomagnetic cell sorting (see above) were pooled and cultured in 96-well plates.

Apoptosis of thymocytes cultured for 10 days with PHA and IL-2 was also evaluated after washing cells with PBS and replating them in fresh RPMI medium without PHA and IL-2, and in the presence of anti-CD38, anti-CD28, anti-CD31 or anti-CD3{epsilon} mAb with or without GAM antiserum. Apoptosis was analyzed by flow cytometry after 24, 48 and 72 h.

In some experiments, thymocytes were co-cultured with L cells (murine fibroblasts) transfected with the human CD31 gene (5:1 ratio) (30,34). Mock-transfected L cells were used as controls. Apoptosis of thymocytes was assessed by flow cytometry after 24, 48 and 72 h co-culture. L cells and thymocytes were distinguished in the flow cytometric assays by their different forward and side scatter properties.

Immunohistochemistry
Antibodies for immunohistochemical analyses were anti-CD38 and anti-CD31. Thymus fragments were snap frozen and embedded in OCT prior to cryostat sectioning. Sections (5-µm) were fixed with ice-cold methanol:acetone (1:1) for 5 min and hydrated with PBS. After saturation of non-specific binding sites with 1.5% BSA in PBS for 1 h, endogenous peroxidase was inactivated by treatment with 0.6% H2O2 in methanol. Samples were incubated overnight at 4°C in a humidified chamber with the specific primary antibody. After washing with PBS, sections were treated with biotin-labeled secondary antibody followed by avidin, according to the streptavidin–biotin PAP method. The site of immunoprecipitate formation in the tissue was demonstrated by incubation with 3,3'-diaminobenzidine, which forms a red/brown reaction product identifiable by light microscopy.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Surface expression of CD38, CD31, CD28 and CD3{epsilon} on human thymocytes
Freshly isolated thymocytes comprise a variety of T cells at different developmental stages. Although a maturational continuum exists, the simultaneous expression of CD4 and CD8, together with CD1a, indicates that the large majority of thymocytes are at the DP stage. Immature precursors and cells at later maturational steps are defined by the lack of CD4 and CD8 expression or, respectively, by the presence of one or the other of these markers, DN and SP cells. To assess the presence of CD38 and CD31 on these subsets, freshly isolated thymocytes comprising >=95% DP cells were considered representative of this cell population, as also shown by CD1a expression. These cells were positive for CD38 and, albeit at lower density, for CD31 (Fig. 1, column 2).

DN cells were negatively selected from freshly isolated thymocytes using anti-CD4- and anti-CD8-coated microbeads. CD4 and CD8 were undetectable, as well as CD1a. CD38 and CD31 were dim on a small fraction of cells (Fig. 1, column 1).

The phenotype of mature thymocytes, SP cells expressing either CD4 or CD8 and lacking CD1a, was analyzed using thymocytes isolated by a two-step immunomagnetic sorting technique (see Methods), or cells stimulated with PHA and cultured for 20 days in the presence of IL-2. Freshly isolated SP cells lost CD1a, a strong indication of maturation (36) (Fig. 1, columns 3 and 4). As shown in Fig. 2(A), thymocytes cultured with PHA and IL-2 for 20 days express CD4 or CD8 and lose CD1a.



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  		Fig. 2. Down-regulation of CD38 on cultured thymocytes. (A) Phenotype of SP thymocytes stimulated with PHA and cultured with IL-2 for 20 days. Note the absence of CD1a expression. (B) CD38 is brightly expressed on freshly isolated thymocytes (T0). Pulse with PHA and culture in the presence of IL-2 leads to down-regulation of CD38 expression after 20 (T20) and 60 (T60) days.

 
A down-regulation of CD38 expression was also observed during maturation of thymocytes stimulated with PHA and cultured with IL-2 for 20 and 60 days (Fig. 2B). CD38 expressed by virtually all freshly isolated thymocytes was detected in 70 and 35% of the cells cultured for 20 and 60 days respectively, and the fluorescence intensity was sharply reduced.

CD28 was expressed by the majority of the thymocytes, although the peak of fluorescence fell into the range between positive and negative cells. We conclude that CD28 is found on the majority of thymocytes at a very low density.

CD3{epsilon} was dim on a small proportion of DN cells, it was bimodal on a large percentage of DP cells and was brightly expressed on virtually all of the SP cells, both CD4+ and CD8+ (Fig. 1).

Localization of CD38 and CD31 in thymus sections, and in TEC cultures
Immunohistochemical analyses showed that CD38 is expressed by the majority of human thymocytes, especially in the cortex of thymic lobules (Fig. 3A). However, some thymocytes in the subcapsular region as well as in the medullary zone were negative (Fig. 3B); this is consistent with phenotypic data indicating that CD38 is undetectable in immature (DN) thymocytes and that it is down-regulated in mature (SP) medullary thymocytes (see Fig. 1).



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  		Fig. 3. Expression of CD38 and CD31 on thymocytes and TEC. (Upper panel) Immunohistochemical localization of CD38 (A and B) and CD31 (C and D) in thymus cryostat sections. (A) Virtually all cortical thymocytes express CD38. Arrow points to an epithelial cell nucleus. (B) At the cortico-medullary (CM) junction some thymocytes (arrows) are negative for CD38. (C) In the subcapsular region (SC) some thymocytes do not express CD31 (arrow). Arrowhead indicates a branching pattern of positivity consistent with a localization of CD31 in the epithelial cell ramifications. (D) Epithelial cells are clustered in the medullary areas of the thymic lobules (M) and are strongly positive for CD31 (arrow). Arrowhead indicates medullary thymocytes that do not express this marker. (Lower panel) A TEC culture (25,26) is slightly positive for surface CD31 (upper row) that is strongly expressed at the cytoplasmic level (lower row). Staining with anti-cytokeratin antibodies confirms the epithelial lineage of the cells.

 
We also investigated the thymic localization of one CD38 ligand, i.e. CD31. This molecule was detected in thymocytes, primarily in the cortex, but also in all of the epithelial components of the tissue, including subcapsular nurse cells, the dendritic cortical epithelium and clusters of medullary epithelial cells (Fig. 3C and D). The presence of CD31 in thymic epithelial cells was also assessed in TEC cultures. These cells expressed surface CD31 dimly and, more markedly, when a cytoplasmic staining was performed (Fig. 3, lower panel). Their epithelial lineage was confirmed by positive staining for cytokeratin (Fig. 3) (25,26).

DP thymocytes are the main population that undergoes apoptosis following CD38 and CD28 ligation and cross-linking
Fresh thymocytes were cultured for 24 and 48 h in the presence of anti-CD38 mAb alone and of its cross-linker, GAM antiserum. An increasing rate of apoptosis was observed in control cultures, particularly after 48 h (from 60 to 33% viable cells) (Fig. 4A and B). The presence of anti-CD38 mAb alone did not affect the rate of thymocyte viability, but its cross-linking by GAM antiserum significantly increased the percentage of apoptotic cells (Fig. 4A and B). IL-2, when included in the assay, had no effects on the rate of thymocyte apoptosis (not shown).



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  		Fig. 4. Cross-linking of CD38 sustains thymocyte apoptosis. Annexin V (x-axis) and propidium iodide (y-axis) staining demonstrates that CD38 cross-linking on human thymocytes leads to their apoptosis. (A and B) CD38, when cross-linked by GAM antiserum, is able to induce an apoptotic signal. Annexin V positivity indicates apoptotic cell death (lower right quadrant), whereas double positivity for propidium iodide and Annexin V (upper right quadrant) corresponds to apoptotic/necrotic cells. (C) SP CD4+ and CD8+ pooled cells do not undergo apoptosis following CD38 cross-linking. These experiments are representative of nine performed with similar results. (D) SP cells were obtained by culturing thymocytes with PHA + IL-2. No apoptosis occurs following CD38 cross-linking. (E) Cross-linking of either CD3 or CD28 alone does not induce thymocyte apoptosis. In contrast, when CD38 or CD38 + CD3 and CD38 + CD28 are cross-linked, significant apoptosis occurs. Note that the highest rate of thymocyte death occurs when both CD38 and CD28 are engaged. This panel is representative of three experiments that yielded similar results. Differences in the experiments shown are due to the fact that distinct thymuses were used in the experiments.

 
In the presence of irrelevant mAb of the same isotype, anti-LAIR1 either soluble or cross-linked (Fig. 4A and B) and anti-CD158b1 (not shown), a rate of apoptosis similar to that of control cultures was determined.

These data suggest that cell death mediated via CD38 affects DP cells predominantly. To support further this contention, DN cells purified as described above were cultured under the same conditions, i.e. in the presence of CD38 ± GAM antiserum. No increase in the rate of apoptosis was detected (not shown).

Freshly isolated SP cells were also analyzed for their susceptibility to undergo apoptosis via CD38. As shown in Fig. 4(C), both engagement of CD38 and its cross-linking had no effect on the rate of thymocyte death. Identical data were obtained using non-pooled SP thymocytes (not shown).

The same result was observed when thymocytes cultured for 20 days with PHA and IL-2 were analyzed (Fig. 4D). It has to be reiterated that these experiments were conducted in the absence of IL-2.

As previously described (14), ligation and cross-linking of CD3{epsilon} alone, as well as of CD28, did not yield significant cell death in any of the thymocyte subsets (Fig. 4E). In contrast, signals generated by cross-linking of both CD38 and CD3{epsilon} or CD28 synergize in inducing apoptosis of DP thymocytes. CD28 is more efficient than CD3{epsilon} in enhancing CD38-mediated apoptosis (Fig. 4E).

A functional role for CD31
As shown in Fig. 3, CD31 is detected in both thymocytes and TEC. In addition to its binding to CD38, CD31 also mediates homotypic adhesive interactions (37). Therefore, it could be responsible for apoptosis that follows CD38 cross-linking in human thymocytes. To address this issue, L cells (murine fibroblasts) were transfected with the human CD31 molecule and mock-transfectants were used as controls. The expression of the CD31 molecule on L cell transfectants was monitored by FACS analysis before co-culture with thymocytes (Fig. 5A). Thymocytes were co-cultured with CD31+ and CD31– L cells, and apoptosis was measured after 24, 48 and 72 h. Data in Fig. 5(B) demonstrate that, in the presence of CD31+ L cells, thymocytes undergo a significantly higher rate of apoptosis (and necrosis) in comparison to co-cultures with mock-transfected cells. This is particularly evident after 48 and 72 h of culture.



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  		Fig. 5. CD31 is the ligand that interacts with thymocyte CD38. (A) Expression of CD31 on L cells transfected with this molecule. (B) Freshly isolated thymocytes were co-cultured with mock-transfected and with CD31-transfected L cells (murine fibroblasts). Thymocytes were subsequently stained as in Fig. 4 for the evaluation of apoptosis and necrosis. In the presence of CD31 transfectants, thymocytes undergo significantly higher levels of cell death. Similar results were obtained in three distinct experiments. (C) No increase of apoptosis occurs when anti-CD31 mAb are cross-linked by GAM antiserum. These data were confirmed by two additional experiments.

 
We next evaluated the effect of anti-CD31 mAb, either soluble or cross-linked, on the death of human thymocytes. No increase of apoptosis occurred when anti-CD31 mAb were cross-linked by GAM antiserum (Fig. 5C). Altogether, these data suggest that CD38–CD31 interactions, rather than a CD31 homotypic ligation, support thymocyte apoptosis

CD38 and CD3{epsilon} co-modulate in the majority of thymocytes that express these markers
We also investigated whether or not CD38 is associated with CD3{epsilon}, as this might enhance its ability to transduce signals.

Although the majority of thymocytes express CD38, as assessed by flow cytometry, immunofluorescence studies employing computer-acquired images indicated that >70% of the thymocytes were CD38+. The discrepancy between this percentage and data from flow cytometry may be due to the lower sensitivity of fluorescence microscopy (see Fig. 1). When modulation experiments were conducted, ~ 60% of the CD38+ cells displayed evidence of modulation (i.e. patching or capping). Among the cells that were modulated for CD38, 40% showed a co-localization of CD3{epsilon} within patches or caps. This indicates that the majority of CD38+ thymocytes display a redistribution of this molecule that goes along with that of CD3{epsilon} (Fig. 6A).



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  		Fig. 6. CD38 and CD3{epsilon} are physically associated on the surface of thymocytes. (A) Capping of CD38 molecules induces co-capping of CD3. By reverting the mAb (i.e. capping for CD3 and staining for CD38) the same result was obtained, although to a lesser extent. The table shows cumulative data from five experiments. The number of caps and co-caps is presented as a percentage of the cells analyzed. Note that, of the two cells shown in these fields, one is completely capped and the other shows patches induced by modulation using both antibodies. (B) No co-capping is observed when anti-MHC class I mAb is used as an irrelevant mAb. Capping of CD38 is not followed by MHC class I redistribution. When MHC class I is capped, CD38 maintains a diffuse localization on thymocytes.

 
When the reverse experiment was conducted, i.e. the CD3{epsilon} molecule was modulated, followed by staining for CD38, we found that ~35% of the cells were positive for CD3{epsilon} by immunofluorescence microscopy. Approximately 28% of these cells formed patches or caps following modulation and, in 12% of the CD3+ cells, evidence of co-modulation for CD38 was detected (Fig. 6A). Control experiments were performed using anti-MHC class I and anti-CD2 mAb. The CD38 molecule was modulated and staining for MHC class I or CD2 followed. None of the cells displayed patches or caps following modulation of CD38 or MHC class I and the immunofluorescence was diffuse in both instances, i.e. MHC class I (Fig. 6B) and CD2 (not shown). The same result was obtained when MHC class I was modulated, followed by staining for CD38 (Fig. 6B).

Altogether, the above data indicate that a large degree of heterogeneity exists among thymocytes, beyond the sharp classification into three distinct developmental stages. Our immunofluorescence studies also show that, in a proportion of thymocytes, CD3{epsilon} and CD38 are physically associated at the plasma membrane level, as they co-modulate following cross-linking of one or the other molecule, and that the mechanism leading to thymocyte death may also involve other molecules, such as CD28, that have been shown to be central to death in the thymus [see also (14,35)].


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
Cell death in the thymus is central to the development and shaping of the repertoire that is responsible for cell-mediated immunity. Positive and negative selection lead to death of the vast majority of developing T cells, and have been attributed to inefficient and to self-reactive antigen receptor expression (2,5). Thymic selection occurs following interaction between maturing T cells with TEC or with cells of hematopoietic lineage, i.e. interdigitating cells and monocytes/macrophages. The latter cells are also responsible for the disposal of apoptotic thymocytes.

The majority of developing thymocytes (>95%) display a DP phenotype, i.e. they co-express both CD4 and CD8 on their surface. It is therefore conceivable that these cells contribute largely to the selective processes and, thus, to intrathymic cell death.

The question addressed to this study is whether interactions between developing thymocytes and accessory cells depend exclusively on cell contacts mediated by MHC (class I and II) expressed by accessory cells and the TCR expressed at variable density on the majority of thymocytes, or else other molecular interactions may favor or hamper the death of thymocytes. It is known that interactions between adhesion molecules expressed by thymocytes and by their counterparts (i.e. ligands present on accessory cells of both epithelial and hemopoietic lineage) may interfere with the selective processes of thymocytes, leading to their death or survival (7–8,23–25).

We show that CD38, expressed by the majority of thymocytes, and one of its ligands, CD31, found at low density on all thymocytes and, virtually, on all of the epithelial cells, sustains thymocyte apoptosis. This indicates that additional molecular interactions between thymocytes and epithelial cells may regulate cell death within the thymus.

As selection of thymocytes occurs mainly at the DP stage, it was worth investigating the effects of CD38/CD31 ligation in this cell subset. We show that CD38 cross-linking by GAM antiserum or by interaction with CD31-transfected fibroblasts significantly increases thymocyte death. It thus appears that other receptor–ligand interactions, besides class I–II MHC–TCR, are relevant for thymic selection.

Since CD38 has a short cytoplasmic tail, several molecules have been described to associate with this receptor. CD16 on NK cells, BCR on B cells and the CD3 complex on Jurkat T cells have been shown to be adaptor proteins that transduce an activation signal mediated by CD38 (35,38–40).

Data from the present study indicate that CD38 and CD3{epsilon} are physically associated on the cell membrane of a proportion of developing thymocytes. CD3{epsilon} co-localizes in patches and caps formed following CD38 modulation, and the same occurs when CD3{epsilon} is modulated first, although to a lesser extent. It thus appears that chains of the CD3 complex function as adaptor proteins when thymocytes are triggered via CD38. A previous report (40) indicates that CD38 is segregated in lipid membrane rafts and that, when lipid rafts are isolated, CD3 is present in the same fractions. This supports our immunofluorescence findings indicating that the two molecules may be associated on the cell membrane. Controls for these data are provided by the finding that CD38 capping is not followed by MHC class I (Fig. 6) and CD2 (not shown), and that MHC class I is modulated independently of CD38 (Fig. 6B).

Although CD3{epsilon} is certainly involved in transducing an apoptotic signal in DP thymocytes as well as in Jurkat cells (39,41), this does not necessarily rule out the involvement of other molecules in this process. We have observed that CD38 co-modulates with CD28 following cross-linking of the latter molecule (not shown) and that, more importantly, CD28 enhances CD38-mediated apoptosis. This suggests a complex molecular mechanism that may lead to thymocyte death following CD38 engagement. In addition, this could explain why CD38 engagement is per se able to induce apoptosis, whereas CD3{epsilon} or CD28 alone do not. At variance, thymocyte apoptosis via CD3–TCR requires CD28 co-engagement (14).

CD38 could play a role in passive apoptosis. This type of programmed cell death requires the presence of surface molecules, such as CD38, that are able to induce apoptosis of all DP thymocytes. The TCR engagement with autologous MHC molecules could rescue MHC-restricted thymocytes by subtracting CD3 molecules to CD38.

It is also worth discussing why CD31 and CD38 molecules, both expressed on developing T cells, do not lead to death of all thymocytes. One possibility is that CD31, expressed at low density on thymocytes, is unable to mediate death signals that would interfere with the selection processes. Alternatively, we suggest that co-receptor molecules on thymocytes may rescue these cells from apoptosis mediated by interaction of CD38 and CD31 expressed by neighboring thymocytes.

Thymocytes are embedded in an epithelial network, the thymic stroma which provides a unique microenvironment for T cell development. Thymic stromal cells are phenotypically heterogeneous and comprise distinct subsets of epithelial cells, mesenchymal cells, and bone marrow-derived macrophages and interdigitating cells. These stromal cells produce extracellular matrix and provide direct cellular contacts that support T cell development. Moreover, epithelial cells secrete a variety of cytokines including IL-1, IL-3, IL-6, IL-7, IL-8, granulocyte colony stimulating factor, granulocyte macrophage colony stimulating factor and transforming growth factor-{alpha} (42–44). These cytokines could be responsible for the partial rescue of thymocytes from apoptosis.

Finally, our study shows that DN and SP thymocytes do not undergo apoptosis via the CD38–CD31 pathway. This is not unexpected as DN, subcapsular, thymocytes represent the proliferation compartment within the thymus and SP medullary thymocytes are the population of mature T cells that seeds into peripheral lymphoid tissues.


   Acknowledgements
 
This work was supported by grants from Associazione Italiana Ricerca sul Cancro, Ministero dell’Istruzione, dell’Università e Ricerca Scientifica and partially by Ministero della Salute (Alterazioni Geniche nelle Leucemie Acute, grant 2001,01,X,000177). A. M. is supported by a fellowship from Fondazione Italiana Ricerca sul Cancro. We are also grateful to Luca Bernava for his help with illustrations.


   Abbreviations
 
DN—double negative

DP—double positive

GAM—goat anti-mouse

PBL—peripheral blood lymphocytes

PHA—phytohemagglutinin

SP—single positive

TEC—thymic epithelial cell


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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