ephrinB signal balance determines the pattern of T-cell ... - Nature

Immunology and Cell Biology (2011) 89, 844–852 & 2011 Australasian Society for Immunology Inc. All rights reserved 0818-9641/11 www.nature.com/icb

ORIGINAL ARTICLE

The Eph/ephrinB signal balance determines the pattern of T-cell maturation in the thymus David Alfaro1,4, Juan Jose´ Munõz2,4, Javier Garcıá-Ceca1, Teresa Cejalvo2, Eva Jimeńez3 and Agustıń G Zapata1 In order to carry out an in-depth study of the roles of EphB receptors in T-cell development and to determine the specific relevance of forward and reverse signals in the process, we established severe combined immunodeficient (SCID) mice chimeras with wild-type (WT) or EphB-deficient bone marrow cells. The obtained results demonstrate that EphB2 contributes more significantly than EphB3 in the control of CD4CD8 (DN)–CD4+CD8+ (DP) progression, and that reverse signals generated in SCID mice receiving EphB2LacZ precursors, which express the EphB2 extracellular domain, partially rescue the blockade of DN cell maturation observed in EphB2-null chimeras. In addition, increased apoptotic DP thymocytes occurring in EphB2 and/or EphB3 SCID chimeras also contribute to the reduced proportions of DP cells. However, EphB2LacZ chimeras do not show any changes in the proportions of apoptotic DP cells, thus suggesting that there is a role for ephrinB reverse signaling in thymocyte survival. The maturation of DP to CD4+CD8 or CD4CD8+ seems to need EphB2 forward signaling and EphB3; a fact that was confirmed in reaggregates formed with either EphB2- or EphB3-deficient DP thymocytes and WT thymic epithelial cells (TECs). The DP thymocyte–TEC conjugate formation was also affected by the absence of EphB receptors. Finally, EphB-deficient SCID chimeras show profoundly altered thymic epithelial organization that confirms a significant role for EphB2 and EphB3 receptors in the thymocyte–TEC crosstalk. Immunology and Cell Biology (2011) 89, 844–852; doi:10.1038/icb.2010.172; published online 18 January 2011 Keywords: EphB; EphrinB; T-cell differentiation; thymus Eph/ephrins are involved in many biological processes, such as morphogenesis of different tissues, cell positioning, cell migration, cell attachment/detachment and cell survival as well as proliferation and differentiation.1–4 They comprise 10 EphA and 6 EphB receptors that bind ephrinA (6 members of glycosylphosphatidylinositolanchored proteins) and ephrinB (3 members of transmembrane proteins), respectively.4 Both Ephs and ephrins signal bidirectionally into Eph-expressing cells (forward signaling) and ephrin-expressing cells (reverse signaling), and exhibit important redundancy as each receptor binds several ligands and vice versa.1,4 Most Eph and ephrins, including EphB2 and EphB3 and their ligands, ephrinB1 and ephrinB2, the molecules studied in this study, have been detected in the thymus, which is the primary lymphoid organ involved in T-cell maturation.5–8 They are expressed in the main thymocyte subsets as well as in the thymic epithelial cells (TECs) of adult and fetal mice.5 In addition, EphB2- and/or EphB3deficient thymuses undergo profound changes in the thymic epithelial network9 and slight changes in the T-cell maturation, thereby largely affecting the CD4CD8 (DN) cell compartment.5 Other in vivo and in vitro studies have demonstrated that altered Eph/ephrin signaling induces important changes in the T-cell differentiation and/or in the TECs.6,10

Despite this ‘weak’ phenotype of mutant thymocytes, it is important to determine the possible role of EphB2 and/or EphB3 on thymocyte maturation, as we recently demonstrated that these molecules govern cell autonomously in the TEC development.11 In this regard, we have comparatively analyzed the T-cell differentiation of either wild-type (WT) or EphB2- and/or EphB3-deficient bone marrow cell progenitors in the thymus of severe combined immunodeficient (SCID) mice, as well as its effects on the organization of the thymic epithelial network; this was evaluated by immunofluorescence. Furthermore, in order to evaluate the importance of forward and reverse signaling in these processes, we studied these same parameters in SCID mice receiving bone marrow cell progenitors that expressed a truncated EphB2, devoid of the cytoplasmic domain.12 EphB2lacZ/lacZ (EphB2LacZ) cells do not receive forward signals through EphB2, but activate reverse signals to neighboring ephrinB-expressing cells. On the other hand, we used reaggregates established with either WT or EphB-deficient CD4+CD8+ (DP) thymocytes and fetal TECs to determine the relevance of EphB2 and EphB3 in the progression of DP cells to mature CD4+CD8 or CD4CD8+ (SP) thymocytes. Finally, we examined the capacity of EphB-deficient DP thymocytes to generate cell conjugates with TECs, as an indirect manner in which to determine their involvement in the attraction/repulsion processes that govern thymocyte–TEC interactions.

1Department of Cell Biology, Faculty of Biology, Complutense University of Madrid, Madrid, Spain; 2Microscopy and Cytometry Centre, Complutense University of Madrid, Madrid, Spain and 3Department of Cell Biology, Faculty of Medicine, Complutense University of Madrid, Madrid, Spain 4These authors contributed equally to this work. Correspondence: Professor AG Zapata, Department of Cell Biology, Faculty of Biology, Complutense University of Madrid, C/Jose´ Antonio Novaís, Madrid 28040, Spain. E-mail: [email protected] Received 28 September 2010; revised 6 December 2010; accepted 20 December 2010; published online 18 January 2011

Eph/ephrinB regulates thymocyte development D Alfaro et al 845

RESULTS Bone marrow precursors isolated from either WT or EphB-deficient (EphB2/, EphB2LacZ, EphB3/ and EphB2/B3/) mice were intravenously injected into immunodeficient SCID mice. The chimeric thymuses (thereafter referred to as WT-SCID or EphB-SCID) were examined 6 weeks later by flow cytometry to analyze changes in both cell numbers and proportions of distinct thymocyte sub-populations. First, we checked the presence of mutant alleles in both the thymus and the bone marrow of chimeric SCID mice. Our results demonstrated that the SCID thymuses were successfully recolonized by deficient precursor cells (Figure 1a). EphB-deficient bone marrow cells mature differentially in the chimeric SCID thymuses SCID thymuses reconstituted with bone marrow precursors isolated from WT mice (WT-SCID) yielded both normal numbers and proportions of the thymocyte subsets, as previously reported by other authors.13 In those studies, it was clearly established that most donor cells differentiate, whereas only a small proportion (range 3–15%) of SCID DN thymocytes did likewise. Contrary to this, SCID mice provided with EphB-deficient bone marrow cells

Figure 1 Chimeric mice, successfully recolonized with the EphB-deficient cells, exhibit an acute thymic hypocellularity. (a) The presence of different mutant alleles was determined by PCR on DNA isolated from total bone marrow cells and the thymocytes of the established SCID chimeras. In both bone marrow and thymus, WT alleles (upper bands), because of the SCID cells, and mutant ones (lower bands), corresponding to the EphB-deficient cells, were found. As a control, DNA isolated from the thymus of CD1 WT mice was used. (b) Bone marrow cell suspensions depleted of mature cells from either WT or EphB-deficient mice were injected in SCID mice and their thymic cellularity was analyzed 6 weeks later. The data shown are means of at least five experiments with s.d. Student’s t-test significance is indicated as *Pp0.05, **Pp0.01 and ***Pp0.005.

(EphB-SCID) exhibited acute thymic hypocellularity (Figure 1b) and remarkable modifications in the proportions of different thymocyte sub-populations (Table 1 and Figure 2). In addition, the phenotypes detected were highly specific, as the recovery of SCID mice with the different defective progenitors (EphB2/, EphB2LacZ, EphB3/ and EphB2/B3/) resulted in different percentages and cell numbers of thymocyte subsets with respect to those values found in WT-SCID mice (Tables 1 and 2 and Figure 2). EphB2/SCID thymuses showed a drastic reduction in the number of total thymocytes (Figure 1b) that coursed with a massive accumulation of the proportions of DN cells and an almost total disappearance of DP thymocytes as well as decreased proportions of mature SP cells, both CD4+CD8 T cell receptor (TCR)ab+ (SP-CD4) and CD4CD8+ TCRab+ (SP-CD8) thymocytes (Table 1 and Figure 2). Moreover, the blockade of DN cell maturation observed in EphB2/SCID mice correlated with increased absolute numbers of DN cells, almost doubled the values of WT-SCID mice, whereas those of both DP and SP thymocytes fell dramatically (Table 2). The SCID mice that received EphB2LacZ progenitors (EphB2LacZ-SCID) exhibited a less severe phenotype with reduced numbers of total thymocytes when compared with the control value, but showed a higher number than those of EphB2/SCID mice (Figure 1b). They recovered the proportion of DP cells, although that of DN cells was still significantly higher and the values of mature SP-CD4 and SP-CD8 were lower than in WT-SCID mice (Table 1 and Figure 2). In absolute terms, an important accumulation of DN cells remained, whereas the numbers of DP thymocytes were significantly lower than in WT-SCID mice and those of mature SP TCRab+ cells underwent a drastic reduction (Table 2). Accordingly, the EphB2LacZ-SCID mice recovered the DP cell compartment in terms of proportions but not in terms of number and were unable to produce mature SP thymocytes properly. Thus, the presence of the extracellular domain of EphB2, which is capable of activating a reverse signal in the neighboring ephrin B-expressing cells, results in a partial rescue of the severe blockade observed at the DN cell stage in EphB2/SCID mice, although it is still unable to accomplish the complete maturation of thymocytes. Changes in the proportions of different thymocyte subsets were less evident in the EphB3/SCID mice. They showed decreased percentage of DP cells and increased proportion of DN cells, although less severe than those observed in EphB2/SCID (Table 1 and Figure 2). Remarkably, EphB3/SCID mice showed an important accumulation of the proportion of mature SP cells (Table 1). In correlation, the proportion of total TCRab-expressing thymocytes doubled control values (Figure 2). However, in absolute terms, the EphB3/SCID chimeras did not show changes in the DN cell compartment, whereas the numbers of both DP and SP thymocytes were reduced (Table 2). Thus, a reduced number of DP thymocytes and/or a blockade of the DN–DP transition were evident in these mice, although less severe than in EphB2/SCID mice. Although slightly more severe than that described in EphB2/ SCID mice, the phenotype of EphB2/B3/ chimeras was remarkably similar. EphB2/B3/SCID mice exhibited an important accumulation of DN cells that resulted in a drastic reduction in the percentage of DP and SP thymocytes, although a low number of mature TCRab+ cells remained in the thymus (Tables 1 and 2 and Figure 2). On the other hand, there was a statistically significant increase in the percentages of TCRgd cells in all the studied mice receiving EphBdeficient cells, but their absolute numbers did not show significant variations. This suggests that EphB defects largely affect the maturation of TCRab cell lineage (Table 3). Immunology and Cell Biology


Table 1 Percentages of CD4/CD8/TCRab thymocyte sub-populations in SCID chimeric mice

WT EphB2/ EphB2LacZ EphB3/ EphB2/B3/

%DN

%DP

%DP TCRabhi

%CD4 TCRabhi

%CD8 TCRabhi

3.36±1.06 87.78±7.18*** 15.29±5.68*

76.37±8.10 1.83±1.39*** 75.30±5.34

4.03±0.43 0.18±0.14*** 1.38±0.10***

14.83±6.49 1.88±1.14*** 2.18±0.50***

3.06±1.41 0.14±0.13*** 0.29±0.12***

11.31±4.06* 89.34±3.77***

54.91±5.10*** 1.15±1.00***

6.94±0.64*** 0.24±0.17***

25.39±3.41** 2.16±0.72***

6.74±2.01* 0.07±0.05***

Abbreviations: DN, CD4CD8; DP, CD4+CD8+; SCID, severe combined immunodeficient; TCR, T cell receptor; WT, wild type. Numbers of used animals: WT, EphB2LacZ and EphB3/ (n¼5); EphB2/ and EphB2/B3/ (n¼7). Student’s t test significance is indicated as *Pp0.05, **Pp0.01 and ***Pp0.005.

Figure 2 Severe alterations in the thymocyte differentiation occur in SCID mice receiving EphB-deficient precursor cells. Percentages of thymocytes defined by the expression of CD4/CD8 cell markers in SCID mice recovered with either WT, EphB2/, EphB2LacZ, EphB3/ or EphB2/B3/ progenitor cells. A representative plot, of at least five independent experiments, is shown.

Table 2 Absolute numbers (106) of CD4/CD8/TCRab thymocyte sub-populations in SCID chimeric mice


DN

DP

DP TCRabhi

CD4 TCRabhi

CD8 TCRabhi

2.89±1.24 4.41±1.20* 6.79±1.81**

65.88±12.30 0.10±0.09*** 36.28±13.55**

3.47±1.30 0.01±0.01*** 0.66±0.25***

12.25±4.06 0.12±0.10*** 1.10±0.48***

2.12±0.71 0.01±0.01*** 0.14±0.11***

2.55±0.82 4.35±0.63*

12.72±2.91*** 0.06±0.05***

1.61±0.44* 0.01±0.01***

5.79±0.70* 0.09±0.08***

1.55±0.46 0.003±0.003***

Abbreviations: DN, CD4CD8; DP, CD4+CD8+; SCID, severe combined immunodeficient; TCR, T cell receptor; WT, wild type. Numbers of used animals: WT, EphB2LacZ and EphB3/ (n¼5); EphB2/ and EphB2/B3/ (n¼7). Student’s t test significance is indicated as *Pp0.05, **Pp0.01 and ***Pp0.005.

Immunology and Cell Biology


Table 3 The percentage, but not the absolute numbers, of TCRcd-expressing cells increases in all chimeric EphB-SCID


TCRgd cells (106)

% TCRgd cells

0.23±0.05 0.25±0.09

0.26±0.04 4.71±0.79***

0.23±0.09 0.20±0.08 0.24±0.09

0.51±0.16* 0.83±0.26* 5.81±1.63***

Abbreviations: SCID, severe combined immunodeficient; TCR, T cell receptor; WT, wild type. Numbers of used animals: WT, EphB2LacZ and EphB3/ (n¼5); EphB2/ and EphB2/B3/ (n¼7). Student’s t test significance is indicated as *Pp0.05 and ***Pp0.005.

Decreased total, DP and SP subset cell content observed in the EphB-SCID thymuses correlates with increased apoptosis To examine possible factors contributing to the thymic hypocellularity and variations in the numbers and/or proportions of thymocytes observed in the SCID chimeras, we evaluated the percentage of both apoptotic and cycling cells that occurred in the different T-cell subsets. The flow cytometry analysis of the percentages of apoptotic (annexin V+/propidium iodide) cells showed increased proportions of apoptotic thymocytes in all the mice receiving EphB-deficient cells, except in those injected with EphB2LacZ progenitors (Figure 3a and Supplementary Figure 2). In the animals receiving EphB2-, EphB3- or EphB2/B3-deficient progenitors, the increase of apoptotic cells mainly affected the DP and SP-CD4 cells with a lower incidence in the SP-CD8 cell sub-population. In the case of EphB2LacZ-SCID cells, although there were slight variations in the proportions of apoptotic cells present in the distinct thymocyte subsets, the differences were not statistically significant with respect to the control values. Likewise, no statistically significant differences were found in the percentages of thymic cycling cells occurring in the EphB-SCID mice (Figure 3b). These results suggest that the decreased numbers of DP thymocytes observed in EphB2 and/or EphB3 chimeras are not only because of a blockade of DN cell maturation, but also the increased proportions of apoptotic DP cells, especially in the case of EphB3/SCID mice that show a less severe blockade of thymocyte maturation. Both EphB2 and EphB3 are necessary for the DP–SP cell progression The described results herein suggest a role for the reverse signal, activated by the EphB2 extracellular domain, in governing the DN–DP transition. Furthermore, EphB2LacZ-SCID mice, which showed normal proportions of total DP thymocytes, were unable to generate normal SP thymocytes; this suggests that EphB2 forward signaling could also be involved in this last step of T-cell maturation. EphB3 could also be implicated in the maturation of SP thymocytes as EphB3-SCID chimeras showed altered proportions of these cells. In order to confirm these findings, we examined the capacity of DP thymocytes isolated from EphB2-, EphB2LacZ- or EphB3-deficient mice to differentiate in reaggregate thymus organ cultures (RTOCs) established with fetal TECs. After 5 days of culture, we determined, by flow cytometry analysis, the proportion of yielded mutant SP thymocytes with respect to what was produced by RTOCs established with WT DP thymocytes (Figure 4). Remarkably, the proportions of thymocytes capable of reaching the SP cell compartment in RTOCs established with EphB-deficient DP thymocytes were significantly lower than in those formed with WT DP thymocytes (Figure 4). These results confirm the involvement of both forward EphB2

Figure 3 Changes in the proportions of apoptotic thymic cells, but not of cycling cells, occur in the chimeras established with EphB-deficient bone marrow cells (except in EphB2LacZ). The data shown are means of at least five experiments with s.d. (a) Note that the increase of apoptotic cells (AnnexinV+/propidium iodide (PI)) largely affected DP and SP thymocytes. The Student’s t-test significance level is indicated as *Pp0.05, **Pp0.01 and ***Pp0.005. (b) Percentage of cycling cells in the total thymic cell population and the different CD4/CD8 cell subsets observed in the studied experimental conditions.

Figure 4 RTOCs established with WT, EphB2/, EphB2LacZ or EphB3/ DP cells and fetal TECs confirm a role for these receptors in the DP–SP cell progression. The proportions of SP thymocytes (both SP-CD4 and SP-CD8) obtained from RTOCs formed with either EphB2/, EphB2LacZ or EphB3/ DP cells, with respect to those yielded by control RTOCs established with WT DP cells, are shown. The data shown are means of at least five experiments with s.d. One-sample Student’s t-test significance level: ***Pp0.005.

signaling and EphB3 receptors in the final differentiation of DP thymocytes to SP cells. Altered capacity of EphB-deficient DP thymocytes to form cell conjugates with TECs As Eph/ephrins are involved in cell attracting/repulsion,14 a key point in the TCR-dependent thymocyte–stroma interactions,15 we analyzed Immunology and Cell Biology


the capacity of WT and EphB-deficient DP thymocytes to form in vitro cell conjugates with WT TECs. The number of cell conjugates formed with WT DP cells peaked after 15 min to gradually decrease later, but the peak of cell conjugates established between EphB-deficient DP and WT TECs occurred earlier, at the 10-min mark. Furthermore, DP cells isolated from EphB2- and/or EphB3-deficient mice generated less than half of the conjugates than the WT ones (Figure 5a), whereas EphB2LacZ DP thymocytes were able to form a higher number of conjugates than the control cells (Figure 5a). Moreover, we microscopically evaluated the accumulation of phospho-tyrosine (pTyr) at the immunological synapse of the formed conjugates, as a way in which to evaluate the involvement of EphB in the TCR signaling activation. In all cases, the conjugates established with EphB-deficient DP cells showed a lower pTyr accumulation than the control ones (Figure 5b). SCID chimeras supplied with EphB-deficient cells show profound alterations in the thymic epithelium organization The data presented suggest that the reverse signal because of EphB2LacZ cell–TEC interaction induces an unknown signal, presumably transmitted from the TECs to the thymocytes, that is apparently sufficient to allow the progression of DN cells into the DP stage. On the other hand, it is assumed that thymocyte–TEC interactions are essential for both T-cell development16 and TEC organization, and we have previously demonstrated that Eph–ephrins are involved in their control.17 It was important, therefore, to study the conditions of the TEC network in the SCID chimeras.

Figure 5 The lack of EphB2 or EphB3 severely reduces the capacity for establishing cell conjugates between DP thymocytes and TECs and alters its activation signaling. (a) The proportions of WT and EphB-deficient cell conjugates are shown. Data are means with s.d. obtained from at least five independent experiments. (b) DP–TEC conjugates formed for 10 min and immunostained, to detect pTyr at the interaction surface, were counted under a confocal microscope. Bars represent the mean number of pTyr+ events, from three independent experiments with 60 conjugates each. Student’s t-test significance level: *Pp0.05, **Pp0.01; ***Pp0.005. Immunology and Cell Biology

As previous results had demonstrated that TECs from SCID thymuses expand and mature after injections of lymphoid cells,16 we examined the possible effects of the lack of EphB on thymocytes upon the thymic epithelium in those SCID chimeras established with either EphB2, EphB2LacZ or EphB3/ thymocytes (Figure 6). SCID mice reconstituted with WT cell progenitors showed a typical thymic cortex consisting of a continuous network of K8+K5 epithelial cells (Figures 6e and i) and well-delimited medullary areas, mostly containing K8K5+ cells (Figure 6a, dotted lines). EphB2-SCID chimeras showed smaller thymuses (Figure 6b) than those established with either EphB2-LacZ (Figure 6c) or EphB3-deficient thymocytes (Figure 6d). They exhibited a flat, less developed thymic epithelial network (Figure 6b) in which small areas devoid of keratin appeared (Figure 6b, arrows; Figures 6f and j, dotted lines). The thymic parenchyma (Figures 6f and j) showed a dense network of TECs with few intermingled thymocytes and numerous K8+K5+ TECs (Figures 6b, f and j, yellow cells). On the contrary, EphB2LacZSCID or EphB3-SCID chimeras exhibited an altered epithelial meshwork with wide areas devoid of keratin-expressing cells (Figures 6c and d, arrows), although Hoechst 33342 staining demonstrated that inside there were numerous nonepithelial cells (Figures 6k and l, asterisks), and medullary epithelial foci of variable sizes (Figures 6c and d, dotted lines). Furthermore, the cortical epithelium was profoundly disorganized, especially in the EphB2LacZ-SCID thymuses (Figure 6g), whereas in EphB3/SCID some short columns of perpendicularly arranged cortical epithelial cells remained, which showed shortened cell processes (Figure 6h). In these mice, the thymic medulla consisted principally of numerous foci, smaller than those of WT-SCID thymuses, of K8K5+ cells (Figures 6c and d, dotted lines). DISCUSSION This study makes use of a chimeric model in which SCID thymuses are colonized by either EphB2- and/or EphB3-deficient bone marrow progenitors or control WT cells to analyze the possible autonomous role of those molecules in thymocyte development as well as the importance of the balance between forward, reverse and bidirectional (forward plus reverse) Eph/ephrinB signals for the process. First, our results indicated that the suppression of different EphB2 and/or EphB3 signals resulted in specific phenotypes, as previously observed in the thymuses of EphB2 and/or EphB3 knockout (KO) mice.5,9 In fact, these results suggest that signaling throughout EphB2 and/or EphB3 and their ligands, ephrins B, is involved in the control of key steps of thymocyte maturation as well as in the survival of different TCRab cell subsets, largely DP thymocytes, but not in that of TCRgd cell lineage (see Table 3). EphB2/SCID chimeras exhibit massive accumulation of DN thymocytes and an almost total disappearance of DP cells (see Tables 1 and 2 and Figure 2). In addition, increased proportions of apoptotic cells, largely DP thymocytes (see Figure 3a and Supplementary Figure 2), contribute to the marked hypocellularity and extremely low numbers and proportions of DP cells observed in these mice. On the contrary, EphB2LacZ-SCID mice that transmit reverse signals to neighboring ephrinB-expressing cells show a less severe phenotype with increased numbers of thymic cells when compared with EphB2/ SCID mice, although they still show reduced values with respect to those of WT-SCID mice (see Figure 1b). In addition, the absolute number of DN thymocytes is still high and that of DP cells is low (see Figure 1b), although the DP cell compartment partially recovers and reaches a normal proportion of cells (see Table 1 and Figure 2). This fact also correlates with the unchanged proportions of apoptotic DP cells (see Figure 3a and Supplementary Figure 2).


Figure 6 The lack of EphB2, EphB3 or the intracellular domain of EphB2 in thymocytes differentially affects the histological organization of thymic epithelium in chimeric SCID thymuses. Thymus cryosections of SCID chimeras established with either WT (a, e, i), EphB2/ (b, f, j), EphB3/ (c, g, k) or EphB2LacZ (d, h, l) cell progenitors were immunostained with anti-K8 cytokeratin (red) and anti-K5 (green) cytokeratin antibodies. Nuclei (blue) were counterstained with Hoechst 33342. The figure shows general views of WT and deficient thymuses (a–d) and details at higher magnifications (e–l). Medullary areas are marked as dotted lines (a–d). Arrows and asterisks indicate K5K8 areas devoid of cytokeratin. Images (a–d) were obtained at 10 magnification and (e–l) are selected areas of representative analyzed pictures obtained at 20 (e–h) and 40 (i–l) magnifications. Bars represent: 200 mm (a–d), 60 mm (e–h) and 25 mm (i–l).

The reverse signals provided by EphB2LacZ progenitors could partially rescue the blockade of DN cell maturation and be involved, therefore, in the control of DN–DP cell progression. They could target the production of survival signals from the ephrinB-expressing cells (presumably TECs but also thymocytes, as suggested by our own previously reported results5) to protect the developing thymocytes from cell death. The relevance of reverse signals for the biological functions of EphB2 was first evidenced in other systems,12,18 and the involvement of Eph/ephrin signaling in the survival and death of thymocytes and peripheral T lymphocytes has already been extensively discussed, both by us and other authors.6,17,19–21 On the contrary, the cell proliferation does not undergo, however, significant variations in the SCID chimeras (see Figure 3b). This result is quite remarkable because reduced proportions of cycling cells were detected in the thymus of EphB2- and/or EphB3-deficient mice.5 There are several possible explanations for the different phenotypes observed in the EphB-deficient mice and the SCID chimeras described in this study that will be discussed later.

EphB3/SCID thymuses support the maturation of all T-cell subsets, although they show an important decrease in the proportion of DP cells (see Table 1 and Figure 2). Nevertheless, in this case, the decrease is also related to the increased proportions of apoptotic DP cells observed in these chimeras (see Figure 3a and Supplementary Figure 2). These results support an additional role for EphB3 in the progression of DN cells to the DP cell stage, apart from the relevance of signals transmitted by EphB2. In supporting this differential role of EphB2 and EphB3 in the thymocyte development, the phenotype of EphB2/B3/SCID is quite similar to that of EphB2/SCID mice (see Tables 1 and 2 and Figure 2). Apparently, the absence of EphB2 in the developing T-cell progenitors causes a very severe phenotype that masks the lack of EphB3. It is difficult, therefore, to conclude on the existence of a certain functional redundancy for the roles of EphB2 and EphB3 in the thymus. EphB2 and EphB3 could be expressed in different cells, rather than be co-expressed in the same ones, and co-act, therefore, in different stages of T-cell development. They could also induce specific activation of different cellular signals, as has been reported in other systems.22 Immunology and Cell Biology


On the other hand, reverse signals are unable to accomplish the final maturation of DP cells, as EphB2LacZ-SCID chimeras show significantly lower numbers and proportions of both SP-CD4 and SP-CD8 cells when compared with the WT-SCID (see Table 1). Directly testing the role played in this process by EphB2 and EphB3 is difficult, given the low numbers of DP thymocytes yielded from the EphB-SCID thymuses. Therefore, we used an in vitro approach to study the capacity of DP cells isolated from EphB2 KO or EphB2LacZ mice to generate mature SP cells in reaggregates formed with fetal TECs. A similar experimental protocol was used to evaluate the involvement of EphB3 in the DP cell maturation as EphB3/SCID mice showed altered maturation of DP thymocytes that resulted in increased proportions of mature SP cells. Our results (see Figure 4) confirm a role for both molecules, EphB2 and EphB3, in the DP–SP progression as reaggregates established with any EphB-deficient DP cells yielded decreased proportions of SP thymocytes. All studied chimeras show, therefore, severe thymocyte phenotypes in contrast to the conditions described for the EphB2 and/or EphB3 KO mice.5 Several possibilities could explain these remarkable results: 1. As already known, Eph/ephrin expression in a unique cell population (the condition of SCID chimeras in which thymic stroma normally express Eph/ephrin, but lymphoid progenitors do not) results in more severe alterations than when there is a total lack of Eph/ephrin in all thymic cell types (as in the EphB2 and/or EphB3 KO mice).23–26 2. On the other hand, in the Eph/ephrin system, both the signal strength and the type of signal quantitatively and qualitatively determine the cell response.3,27,28 In the WT thymus, where both TECs and developing thymocytes express EphB2 and EphB3, as well as their main ligands, ephrinB1 and ephrinB2,5 cell behavior must be determined by the total balance of signals. This general pattern is obviously different in the KO mice and in the SCID chimeras and could determine changes in the processes of cell attraction/repulsion29 occurring between TECs and thymocytes, thereby resulting in a different, more severe, phenotype in the chimeras than in the KO mice. 3. Finally, some authors have pointed out that an additional source of differences could be the genetic background of the animals.12,30,31 In summary, these results demonstrate an autonomous role for EphB2 and EphB3 in thymocyte development, which participate in controlling the progression of DN cells to the DP cell compartment and subsequently to mature SP thymocytes. In this respect, the comparative analysis of T and B lymphocytes in the peripheral lymphoid organs (mesenteric lymph nodes, spleen and blood) of WT and EphBdeficient chimeras demonstrated that the numbers of peripheral T cells seem to reflect those found in the thymic SP compartment. Thus, there were significant decreases in T-cell numbers in the peripheral organs of EphB2-, EphB2LacZ- and EphB2/B3-SCID chimeras and normal values in EphB3 ones (Supplementary Figures 1A, C and E). It is important to remark that apart from migration from thymus, survival, proliferation and differentiation, which determine the homeostasis of peripheral lymphocytes, they are also dependent on other numerous in situ factors. On the other hand, in all EphB-deficient chimeras, the numbers of peripheral B lymphocytes significantly diminished with respect to control values (Supplementary Figures 1B, D and F). Furthermore, the immunohistochemical analysis of the thymic epithelial network in the chimeric thymuses also shows a nonautonomous role of these molecules in the thymic stroma, which Immunology and Cell Biology

confirms the previously suggested role of Eph/ephrins in the positioning and mutual interactions between TECs and thymocytes.10,17 Thus, SCID chimeric mice that receive EphB2LacZ cells or EphB3/ precursors exhibit important alterations in the histological organization of the thymic epithelium network in both cortex and medulla. The absence of the cytoplasmic domain of EphB2 or the lack of EphB3 in the injected progenitor cells profoundly affects that organization. Although some of the alterations bring to mind the epithelial phenotype observed in the thymuses of EphB2 and EphB3 KO mice,9 they are again specific to the injected lymphoid progenitors. On the other hand, the analysis of EphB2-SCID thymuses reveals that the low number of thymocytes that they contain produces an impaired development of the epithelial network with increasing numbers of K8+K5+ cells, as previously reported in other severely hypoplastic thymuses.32 The role of Eph/ephrinB in the thymus could be therefore mediated throughout the interactions established within the thymus between the developing T-cell progenitors and the epithelial cells of the thymic stroma. We have showed that the in vitro EphB2-Fc or ephrinB1-Fc cell treatment decreased the formation of conjugates formed by DP thymocytes and TECs.10,17 Accordingly, we examined the relevance of EphB2 and EphB3 in thymocyte–TEC interactions by analyzing the capacity to form cell conjugates of either WT or EphBdeficient DP thymocytes with WT TECs and its subsequent activation, by measuring the accumulation of pTyr in the immunological synapse of the conjugates. The numbers of established conjugates decreased when using EphB2- and/or EphB3-deficient DP thymocytes, but they increased with the EphB2LacZ ones—the unique chimeric mice that show normal proportions of DP thymocytes. On the other hand, in all cases, the conjugates formed with EphB-deficient DP thymocytes show lower levels of pTyr than the control ones, established with WT DP cells. These results suggest that Eph/ephrinB signaling is involved in DP– TEC interactions and its subsequent activation. Similarly, the effects mediated by EphB2 and EphB3 in these processes are specific and non-redundant because the absence of EphB2 is not restored by the presence of EphB3 (or any other EphB) and vice versa. Furthermore, the extracellular region of EphB2, expressed on EphB2LacZ DP thymocytes, could contribute to generating attraction signals for the conjugate formation even more efficiently than the whole EphB2 molecule expressed on WT DP cells. It is also possible that the cytoplasmic domain of EphB2 could affect the capacity to form cell conjugates, especially in the early phase of interaction (first 15 min) when the differences between WT and EphB2LacZ DP are more evident. On the other hand, although EphB2LacZ-expressing DP thymocytes efficiently generate cell conjugates, they show decreased levels of pTyr residues; this suggests that both processes are not fully associated or that the reverse signaling generated by these DP cells is not sufficient to fully activate the thymocyte, which results in an immature rather than mature immunological synapse. In summary, these results support the relevance of a well-regulated balance of different Eph/ephrin signals for mediating fine tuning interactions that have been pointed out as being necessary for the correct thymocyte progression throughout the different thymic epithelial microenvironments.33 METHODS Mice EphB-deficient mice, including EphB2/, EphB3/, EphB2/B3/ and EphB2LacZ mice, were provided by Dr Mark Henkemeyer (University of Texas, Southwestern Medical Center, Dallas, TX, USA). EphB2LacZ cells express a truncated form of EphB2 whose cytoplasmic domain has been substituted by the lacZ gene product.12 SCID mice were purchased from Harlan

Eph/ephrinB regulates thymocyte development D Alfaro et al 851 (Harlan Ibe´rica, Barcelona, Spain). All animals were maintained in specific pathogen-free conditions in the facilities at the Complutense University of Madrid and were used in accordance with the animal care protocols approved by this university. At least five homozygous animals for each experimental group, descendents of heterozygous parents, were used in all cases.

Reconstitution assays Bone marrow cell suspensions were harvested from mouse hind limbs and enriched in a Histopaque-1077 (Sigma-Aldrich, St Louis, MO, USA) density gradient following the supplier’s instructions. The cells obtained were then washed in RPMI-1640 (Sigma-Aldrich) with 5% fetal calf serum, stained with an allophycocyanin-conjugated lineage antibody cocktail, Lin (CD3, CD11b, CD45R/B220, Ly-76, Ly-6G, Ly-6C) (BD Biosciences, Erembodegem, Belgium), for mature cell populations, and incubated with anti-allophycocyanin Microbeads (Myltenyi Biotec, Bergisch Gladbach, Germany). Immature cells were negatively isolated using an AutoMACS (Myltenyi Biotec) according to the supplier’s instructions, obtaining a purity of 98–99%. A total of 2.5106 cells were intravenously injected in the tail vein of SCID mice, and after 6 weeks, their organs were analyzed.

PCR analysis Isolation of DNA from thymuses and bone marrow cell suspensions was carried out with Tri-Reagent (Sigma-Aldrich) according to the manufacturer’s specifications. Oligonucleotide sequences and PCR conditions were provided by Dr Henkemeyer for EphB2, EphB2LacZ and EphB3 mutant alleles.12,31 The amplification products were resolved in 1.5% agarose electrophoresis gel.

Flow cytometry Thymocyte suspensions from reconstituted SCID mice were stained for 20 min in phosphate-buffered saline/1% fetal calf serum with specific antibodies against CD4, CD8a, TCRab, TCRgd (BD Biosciences), labeled with either fluorescein isothiocyanate (FITC), phycoerythrin, Tricolor or allophycocyanin, and/or AnnexinVFLUOS and propidium iodide for cell death analysis (Roche Diagnostics, Penzberg, Germany). Lymphocyte suspensions from spleen and peripheral blood, after erythrocyte lysis, and mesenteric lymph nodes, were stained for 20 min in phosphate-buffered saline/1% fetal calf serum with specific antibodies: TCRab FITC and CD19 allophycocyanin (BD Biosciences). They were then washed and re-suspended for analysis. For cell cycle analysis, after surface labeling, cells were fixed in Cellfix (BD Biosciences) overnight and stained with Hoechst 33342 (Molecular Probes, Eugene, OR, USA) in EtOH 30% in phosphate-buffered saline/1% bovine serum albumin for 1 h at room temperature. At least 20 000 cells per sample were analyzed using a FACSCalibur or LSR (BD Biosciences, San Jose, CA, USA) and the CellQuest software at the Microscopy and Cytometry Centre (Complutense University, Madrid, Spain). The significance of the Student’s t-test probability is indicated as follows: *Pp0.05, **Pp0.01 and ***Pp0.005.

Microscopy For immunofluorescence, cryosections from recovered thymuses were fixed in acetone for 10 min and air dried. Slides were stained with anti-mouse keratin 5 rabbit antiserum (Covance, Berkeley, CA, USA) and/or anti-mouse keratin 8 monoclonal antibody Troma-1 (Developmental Studies Hybridoma Bank, Iowa City, IA, USA). After washing, staining was revealed with anti-rabbit IgGAlexa-488 and anti-rat IgG-Texas Red (Jackson ImmunoResearch Europe, Suffolk, UK), respectively. Cell nuclei were detected by staining with Hoechst 33342 (0.2 mg ml–1; Molecular Probes). Sections were mounted with Prolong Gold medium (Invitrogen, Eugene, OR, USA), visualized in a Zeiss Axioplan microscope (Zeiss, Go¨ttingen, Germany; objectives: 10 N/0.30 PlanNeofluar, 20 N/0.50 Plan-Neofluar, 40 N/0.17 Plan-Neofluar) photographed with a Diagnostic SPOT 2 camera (Burroughs, MI, USA), imported with Metamorph 6.0 (Molecular Devices, Chicago, IL, USA) and analyzed with Adobe Photoshop CS3 at the Microscopy and Cytometry Centre.

Preparation of thymocytes, TECs and RTOCs DP thymocytes, at a preselection stage (CD3 negative/low), were obtained either from WT or EphB-deficient mice. After labeling with anti-CD3-FITC, the

CD3+ cells were magnetically depleted using anti-FITC Microbeads (Myltenyi Biotec) and an AutoMACS (Myltenyi Biotec). Flow cytometric analysis of the resulting cell population showed that there were over 95% of DP cells. TECs were obtained by disaggregating 15-fetal day CD1 WT thymus lobes depleted of lymphoid cells by 2-deoxiguanosine treatment, as previously described.17 The resultant suspension was mainly constituted by epithelial cells.34 For RTOCs, DP thymocytes and TECs were mixed together by centrifugation in a 1:1 ratio and the pellets were transferred to 0.8 mm filters.35 After 5 days of culture, the maturation of yielded thymocytes was evaluated by flow cytometry and statistically analyzed with the one-sample Student’s t-test (**Pp0.01 and ***Pp0.005).

Formation and analysis of thymocyte–epithelial cell conjugates Formed thymocyte–epithelial cell conjugates were evaluated by flow cytometry as described previously.36 Briefly, thymocytes labeled with PKH26 (SigmaAldrich) and TECs stained with carboxyfluorescein succinimidyl ester were mixed by centrifugation in a 1:1 proportion and incubated at 37 1C. Analysis of cell pellets immediately following centrifugation was designated time 0. Some unstained conjugates were fixed for 10 min in 2% paraformaldehyde, washed and stained with an anti-pTyr antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). They were mounted with Prolong Gold medium (Invitrogen) and analyzed for pTyr accumulation at the immunological synapse, as described previously,17 in a Leica TCS SP2 (Leica, Heidelberg, Germany) confocal microscope.

CONFLICT OF INTEREST The authors declare no conflict of interest.

ACKNOWLEDGEMENTS We thank Dr Mark Henkemeyer for providing EphB-deficient mice and the Microscopy and Cytometry Centre of the Complutense University for the use of their facilities and technical assistance. We also thank the ‘Developmental Studies Hybridoma Bank’ of Iowa University for supplying the anti-K8 keratin antibody. This work was supported by Grants BFU 2004-03132 and BFU 200765520 from the Spanish Ministry of Education and Science; RD06/0010/0003 from the Spanish Ministry of Health and Consumption and S-BIO/0204/2006 and GR74/910552/2007 from the Regional Government of Madrid.

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