During their final maturation in the medulla, semimature single-positive (SP) thymocytes downregulate activation markers and subsequently exit into the periphery. Although semimature CD4+ SP cells are sensitive to negative selection, the timing of when negative selection occurs in the CD8 lineage remains elusive. We show that the abundance of terminally matured CD8+ SP cells in adult thymus is modulated by the genetic background. Moreover, in BALB/c mice, the frequency of terminally matured CD8+ SP cells, but not that of CD4+ SP cells present in thymus, varies depending on age. In mice lacking expression of the adhesion receptor CD155, a selective deficiency of mature CD8+ SP thymocytes was observed, emerging first in adolescent animals at the age when these cells start to accumulate in wild-type thymus. Evidence is provided that the mature cells emigrate prematurely when CD155 is absent, cutting short their retention time in the medulla. Moreover, in nonmanipulated wild-type mice, semimature CD8+ SP thymocytes are subjected to negative selection, as reflected by the diverging TCR repertoires present on semimature and mature CD8+ T cells. In CD155-deficient animals, a shift was found in the TCR repertoire displayed by the pool of CD8+ SP cells, demonstrating that CD155 is involved in negative selection.

Among hematopoietic cells, T cells are unique because they accomplish their development in thymus and not in bone marrow. To achieve this, thymic precursors enter the organ at the cortico-medullary junction and start a differentiation program (1). This is connected to an intrathymic migration path guiding the developing cells through the cortex and subsequently into the medulla (2). The early cortical steps encompass the double-negative stages 1–4 prior to the onset of CD8 expression, giving rise to immature single-positive (SP) cells (1). Shortly thereafter, CD4 expression commences, yielding double-positive (DP) thymocytes that are subjected to positive selection (3). Cells successfully passing positive selection develop into semimature CD4+ or CD8+ SP cells, a step that is believed to be associated with their transit into the medulla. Although negative selection eliminating self-reactive SP cells by apoptosis can take place to a certain extent in the cortex (4), it is generally assumed that these processes are mediated by medullary epithelial cells (mTECs) as well as dendritic cells (DCs) amply present in the medulla (5, 6). Particularly DCs, but also mTECs, express costimulatory molecules as well as a broad array of self-Ags required to eliminate self-reactive T cells (5, 7). According to a redundancy model, many costimulatory molecules (e.g., CD5, CD28, CD40, CD43, and CD152), as well as the chemokine receptor CCR7 are capable of participating and cooperate in negative selection (811). In contrast, little is known about the involvement of adhesion receptors in this process, although evidence exists that functional interference with integrins/ligands or cadherins causes a perturbed genesis of thymocytes at several stages of their development (1214). During their final maturation, SP cells downregulate activation markers, such as CD69 and CD24, and upregulate CD62L (15, 16), rendering cells competent for emigration. Egress of matured naive SP cells crucially depends on the expression of the sphingosine 1-phosphate (S1P) receptor S1P1. Cells lacking S1P1 or treated with FTY720, a drug interfering with S1P receptor-mediated activity, are blocked in emigration (1719).

The adhesion receptor CD155 was originally identified as the cellular receptor for poliovirus, representing the major factor determining the host and tissue tropism of the virus (20). CD155 belongs to the nectins, a family of Ig-like transmembrane proteins (21). All known physiological and pathogenic functions of CD155 are mediated via its outermost V-like domain harboring the docking epitopes for its known ligands: nectin-3, vitronectin, CD226, CD96, and poliovirus (2229). CD155 is involved in the establishment of adherens junctions between endothelial and epithelial cells (21) and was shown to influence cell motility, migration, and proliferation (30, 31). In addition, CD155 is overexpressed in several tumor types, an observation that explains the susceptibility of tumors to NK-driven eradication along the CD226/CD155 axis (3234). However, the latter interaction is also relevant for NK-mediated elimination of DCs; thus, it may contribute to shape the DC pool that is able to induce T cell responses (35, 36). Moreover, we demonstrated recently that CD155 is implicated in the generation of a humoral immune response upon oral ingestion of Ags (37). This may be explained by the finding that CD155-deficient mice harbor reduced numbers of follicular helper T cells in their Peyer’s patches (38).

In this study, we show that the lack of CD155 provokes a shift in the TCR repertoire displayed by CD8+ SP thymocytes. We also observed a significant reduction in the number of fully matured CD8+ SP cells in adult thymi, whereas the frequency of semimature CD8+ SP cells is largely unaffected. Interestingly, mature CD8+ SP cells seem to accumulate in an age-dependent fashion in wild-type (WT) BALB/c thymus, offering an explanation for why the paucity of CD8+ SP cells triggered by CD155 deficiency starts to emerge in adolescent mice. Because we failed to observe defects in maturation and the degree of emigration or apoptosis regarding CD155-deficient CD8+ SP thymocytes, our findings suggest that newly matured CD8+ thymocytes cannot be retained in the medulla when CD155 expression is missing. This demonstrates that the adhesive mechanisms keeping SP cells inside the medulla differ between CD4+ and CD8+ SP cells, semimature and mature CD8+ SP cells, and mature CD8+ SP cells produced in young versus adult mice.

WT BALB/c (H2d) and C57BL/6 (H2b) mice were purchased from Charles River Laboratories (Sulzfeld, Germany) or bred in Hannover Medical School’s animal facility. DBA/1 (H2q), C3H (H2k), and SJL (H2s) mice were purchased from Janvier (Le Genest St. Isle, France). CD155 knockout (KO) mice were described before (37). Mice were crossed onto the BALB/c background for at least six generations. All experiments including animals were conducted according to the regulations of the local government and institutional guidelines.

Single-cell suspensions of thymi were obtained by mincing the organs through a cell strainer dish. Cells were washed in FACS buffer (PBSd/2% FBS), counted, and stained in 96-well plates, according to standard procedures, in 50 μl reaction volume. After three final washes, DAPI was added to exclude dead cells from analysis. The Annexin-V-Fluos kit (Roche, Basel, Switzerland) was used to analyze apoptosis.

The following Abs were used: Vβ 2, 3, 4, 5, 6, 7, 8, 11, and 12 TCR (BD Biosciences, San Jose, CA); CD24-FITC, CD69-PerCp-Cy5.5, CD45.2-PerCp-Cy5.5, Ly51, and SA-PerCP (all from BD Biosciences); CD25-Alexa Fluor 488, CD44-Alexa Fluor 405, CD44-PE, CD44-APC, CD11b-PE, DX5-bio, αβTCR, and SA-Pacific Orange (all from Invitrogen, Karlsruhe, Germany); CD117-PE; (eBioscience, Frankfurt, Germany); EpCAM-PE (eBioscience); and CD24-PE-Cy7 (Biolegend, Uithoorn, The Netherlands). The following homemade Abs were used: CD3-bio (17A2), CD4-bio (RMCD4-2), CD8β-bio (RMCD8-2), CD19 (1D3), TER119, GR1, γδTCR-bio (GL3), CD62L (MEL-14), B220 (TIB146), CD155 (4G3), and CD226 (3B3); they were either used directly labeled as indicated or detected with fluorochrome-labeled standard secondary Abs. Flow cytometry was performed on an LSRII flow cytometer (BD Biosciences); the data were evaluated using WinList 5.0.

WT BALB/c mice were injected i.v. with 400 μg anti-CD155 mAb 4G3 (37) or an isotype control mAb every 5 d. The animals were sacrificed 28 d following the first injection.

Mice were administered sterile water or FTY720 (40 μg/mouse; Calbiochem, San Diego, CA) in drinking water or by oral gavage every 3 d. Ten to 12 d later, mice were sacrificed and analyzed. Drug effectiveness was confirmed by analyzing lymphopenia induction in the blood of treated animals (data not shown).

Animals were anesthetized, and the chest was disinfected with 70% ethanol. An ∼5-mm longitudinal incision was made into the skin directly above the sternum, then the area was cleaned with 70% ethanol by wiping carefully with a lint-free cloth, followed by scanning the sternal/rib area for proper injection (the thymus is located underneath the second rib, close to the sternum). A 27G needle was used to inject 10 μl 1 mg/ml FITC solution (Sigma-Aldrich, St. Louis, MO) per lobe. The injection deepness was ∼5 mm. The skin incision was closed with surgical clips. The spleen and lymph nodes were harvested for analysis ∼40 h postinjection.

To enable the analysis of bone marrow chimeras at an age when thymus development culminates, newborn mice no older than 1 d were subjected to two irradiations of 2.5 Gy each, separated by 4 h, followed by bone marrow transfer. Bone marrow was harvested from donor mice and depleted for differentiated cells by applying a lineage mixture of rat anti-mouse Abs recognizing Ter119, CD4 (RMCD4-2), CD8β (RMCD8-2), CD11b (M1/70, eBioscience), CD19 (1D3), DX5 (Biolegend), and GR1, followed by incubation with sheep anti-rat IgG coupled to Dynabeads (Invitrogen). Magnetic separation was performed according to the manufacturer’s instructions. Lineage negative cells were concentrated in PBS to allow intrahepatic injection of 2 × 105 cells in a total of 25 μl. Mice were analyzed 8–10 wk following the bone marrow transfer. All animals exerted normal behavior, yet they were distinguished by a substantially reduced weight compared with age-matched controls.

Cells were prepared from thymi pooled from five WT or CD155-deficient mice. CD8 SP cells were enriched by negative selection, removing CD4+ cells via anti CD4-biotin/M-280 Streptavidin Dynabeads (Invitrogen).Then staining with anti CD8α-Cy5/CD24-PECy7 was done, followed by flow cytometric sorting into CD8+CD24hi and CD8+CD24lo fractions. Successful separation was controlled by reanalysis. Up to 5 × 105 cells were used for RNA isolation (Absolutely RNA Microprep kit, Stratagene, La Jolla, CA); subsequently, synthesis of the first strand of cDNA (Superscript II reverse transcriptase; Invitrogen) was done using random hexamer primers. The expression of GAPDH and S1P1 was analyzed using a Lightcycler 2.0 (Roche) and the Fast Start DNA Master plus SYBR Green Kit (Roche) or the SYBR Premix Ex Taq Kit (Takara Shuzo, Otsu, Japan). The primers applied and the relative comparative quantification of expression levels was done based on GAPDH expression, as described earlier, except that a cloned PCR product representing the GAPDH amplicon was used for standardization (39).

In our earlier analyses of CD155-deficient mice, we noticed regular numbers of peripheral T cell subsets in all secondary lymphoid organs investigated (37). Moreover, we also failed to observe any differences in size or obvious anatomical and morphological aberrations in the lymphoid organs (37). When investigating CD155-deficient thymi of adult BALB/c mice (8–10 wk) by immune histology, regularly sized and distributed cortico-medullary areas were detected (not shown). In addition, determination of overall thymic cell counts failed to reveal significant differences compared with WT mice. However, when analyzing the cellular composition of thymocytes by flow cytometry, we found a significant reduction in the frequency of CD8+ SP cells in mutant thymi (Fig. 1A; WT: 5.08% ± 1.06%; KO: 3.23% ±0.60%), which was paralleled by a slight increase in the percentage of DP cells (data not shown). In contrast, the frequency of CD4+ SP cells remained unchanged (WT: 10.28% ± 1.17%; KO: 9.39% ± 1.50%). A considerable proportion of the flow cytometric determinations of CD8+ SP frequencies were also done more stringently, including markers revealing other cell types that are present in the thymus, and therefore, may contribute to the observed imbalance. However, when gating was done to exclude memory cells (CD44hi), regulatory T cells (CD25+), TCRγδ T cells (GL3+), and NK T cells (CD3+DX5+), the same degree of discrepancy between WT and KO thymocyte numbers was found, confirming that regular αβTCR CD8+ T cells are affected by CD155 deficiency (data not shown).

FIGURE 1.

Lack of CD155 causes a deficiency in mature CD8+ thymocytes. A, Flow cytometric analysis of thymocytes and peripheral T cells of WT and CD155−/− origin showing the separation according to CD4 and CD8 expression. Dead cells (DAPI) and doublets were excluded. For peripheral cells (pLNs), a pregate was set to include DAPICD62L+CD3+ cells only. The lower panels depict the percentages of CD4+ SP and CD8+ SP cells. Each circle represents one animal. Closed circles represent WT mice; open circles denote CD155-deficient mice. B, Distribution of SP cells and peripheral T cells in mice treated with anti-CD155 mAb. Closed circles and open circles represent untreated and mAb-treated mice, respectively. ***p < 0.001. C, Left panels: CD24 expression of CD8+ SP and CD4+ SP cells from WT and CD155-deficient thymus as given in A. Shown are representative graphs as overlays of WT and KO cells. Right panel: Expression of S1P1 was determined in FACS-sorted populations as indicated. Expression is given in relation to GAPDH. Results from two independent experiments are shown; runs were done in duplicate. Error bars depict SD.

FIGURE 1.

Lack of CD155 causes a deficiency in mature CD8+ thymocytes. A, Flow cytometric analysis of thymocytes and peripheral T cells of WT and CD155−/− origin showing the separation according to CD4 and CD8 expression. Dead cells (DAPI) and doublets were excluded. For peripheral cells (pLNs), a pregate was set to include DAPICD62L+CD3+ cells only. The lower panels depict the percentages of CD4+ SP and CD8+ SP cells. Each circle represents one animal. Closed circles represent WT mice; open circles denote CD155-deficient mice. B, Distribution of SP cells and peripheral T cells in mice treated with anti-CD155 mAb. Closed circles and open circles represent untreated and mAb-treated mice, respectively. ***p < 0.001. C, Left panels: CD24 expression of CD8+ SP and CD4+ SP cells from WT and CD155-deficient thymus as given in A. Shown are representative graphs as overlays of WT and KO cells. Right panel: Expression of S1P1 was determined in FACS-sorted populations as indicated. Expression is given in relation to GAPDH. Results from two independent experiments are shown; runs were done in duplicate. Error bars depict SD.

Close modal

We recently established a panel of anti-CD155 mAbs; two of these (3F1 and 4G3) block the interaction of CD155 with its ligands CD96 and CD226 [(37, 40) and data not shown]. Therefore, mAb 4G3 was injected repeatedly into WT BALB/c mice for 4 wk, and the thymi and the peripheral lymph nodes (PLNs) were analyzed. mAb 4G3 was able to provoke a loss of CD8+ SP cells to an extent seen in CD155-deficient mice (Fig. 1B). The frequencies of peripheral T cells were not influenced by mAb treatment, demonstrating that T cells were not depleted. This suggested that the observed deficiency in CD8+ SP cell numbers can be triggered instantaneously by interrupting CD155 adhesion.

We next sought to characterize the CD8+ SP thymocytes in more detail with regard to their maturation stage. It is known that SP cells downregulate CD69 and CD24 during their final maturation. CD24 staining revealed a characteristic distribution pattern among WT CD8+ SP cells. The narrow majority of cells displayed intermediate levels of CD24, representing semimature CD8+ SP cells. A considerable proportion of cells was CD24lo (mature CD8+ SP cells), whereas a minor fraction was CD24hi (Fig. 1C). The latter population is composed of immature SP cells (TCRαβ negative, data not shown), which develop into DP cells. When comparing this pattern with that obtained from CD155-deficient cells, it was evident that the lack of CD155 almost exclusively affects the most mature CD24lo cells because, to a large extent, these cells were missing in the KO thymi (Fig. 1C). The CD24-staining profiles of the CD4+ SP WT and mutant cells did not diverge significantly from each other, in accordance with the finding that the cell frequencies were identical. As mentioned above, injection of anti-CD155 mAb 4G3 caused a deficiency of thymic CD8+ SP cells. Analysis of the CD24 expression profile of mAb-treated CD8+ SP cells found that they displayed the same phenotype as seen in the classical KO cells, with the mature CD24lo cells almost completely missing in the pool of CD8+ SP thymocytes (data not shown).

Mature CD4+ SP thymocytes upregulate their S1P1 expression, rendering them competent for emigration (19). Therefore, we investigated the expression of this receptor by real-time PCR in semimature and mature CD8 SP cells separately (Fig. 1C). In both, WT and CD155 mutant CD8+ SP S1P1 expression was virtually absent in semimature cells. A pronounced upregulation of S1P1 expression was observed in the mature subpopulation to an extent that did not differ between WT and CD155-deficient cells. This indicates that the lack of mature CD8+ SP cells in CD155-deficient thymi is not caused by an aberrantly regulated S1P1 expression.

In general, C57BL/6 thymi harbor a lower percentage of CD8+ SP cells compared with the BALB/c background, indicating that the abundance of SP cells varies among different mouse strains. When analyzing the cellular composition of the CD8+ SP cell pool, it was evident that only a small proportion of mature CD8+CD24lo cells is present in adult thymus of C57BL/6 mice (Fig. 2A). We selected additional inbred mouse strains of differing MHC class I haplotypes (see 1Materials and Methods) and investigated the composition of their CD8+ SP cell populations in more detail. To this end, the expression analysis of the CD155 counter-receptor CD226 was included. CD226 is expressed in semimature and mature, but not immature, CD8+ SP cells, thus allowing a better discrimination of immature and semimature CD8+ SP cells (36). In addition, CD226 expression declines in mature CD8+ SP cells of BALB/c origin. By and large, the investigated strains fall into two categories: DBA/1, SJL, and BALB/c mice possess considerable amounts of mature CD8+ SP cells in their thymi as illustrated by their low ratio of semimature to mature CD8+ SP cells, whereas C3H and C57BL/6 mice largely lack that cell type (Fig. 2B, 2C). In addition, a characteristic CD226 expression pattern could be found in each strain, with SJL CD8+ SP cells expressing this CD155 counter-receptor at a low level and DBA/1 mice exhibiting the biggest difference in CD226 expression between semimature and mature CD8+ SP cells. This is an interesting observation considering that BALB/c thymocytes express slightly more CD155 than C57BL/6 thymocytes (data not shown). We analyzed heterozygous BALB/c animals to test whether the level of CD155 expression is critical for modulating the abundance of mature CD8+ SP cells. CD155 is expressed in a gene dosage-dependent fashion, resulting in half-maximum CD155 levels on CD155+/− cells (data not shown). However, CD155+/−CD8+ SP cells resembled their WT counterparts, whereas the CD155−/−CD8+ SP cells of BALB/c origin mimicked the CD57BL/6 WT phenotype with regard to the ratio of semimature to mature CD8+ SP cells (Fig. 2C).

FIGURE 2.

Abundance of mature CD8+ thymocytes is strain dependent. A, Flow cytometric analyses of CD8+ SP cells are shown as in Fig. 1C for the strains as indicated. B, Refinement of the analysis seen in A including CD226. A and B show representative graphs and scatter plots, whereas the results obtained by gating according to B are summarized in C. The ratios of semimature/mature CD8+ SP cells are shown. Each dot represents one thymus. Het, heterozygous CD155+/− BALB/c animals; ISP, immature SP.

FIGURE 2.

Abundance of mature CD8+ thymocytes is strain dependent. A, Flow cytometric analyses of CD8+ SP cells are shown as in Fig. 1C for the strains as indicated. B, Refinement of the analysis seen in A including CD226. A and B show representative graphs and scatter plots, whereas the results obtained by gating according to B are summarized in C. The ratios of semimature/mature CD8+ SP cells are shown. Each dot represents one thymus. Het, heterozygous CD155+/− BALB/c animals; ISP, immature SP.

Close modal

All data presented in the following sections refer to BALB/c mice.

Thymus represents a highly dynamic organ that grows tremendously in size after birth. At ∼3 wk of age, thymi have grown to their maximal size, which remains constant during the following weeks until atrophy begins (41). When analyzing the emergence of SP cells in WT BALB/c thymus, we found that the frequency of CD4+ SP cells reached a plateau at ∼3 wk of age, concurring with thymic growth dynamics (Fig. 3A). In contrast, expansion of the CD8+ SP cell pool was delayed (peaking at ∼6–8 wk; Fig. 3B), confirming earlier reports (41). Considering this, it was of interest to monitor the progression of the observed CD8+ SP phenotype, caused by CD155 deficiency, from suckling to advanced adulthood that is distinguished by thymus involution. The frequencies of CD155-deficient CD4+ SP cells did not differ significantly from those of WT cells at any of the ages investigated (Fig. 3A). In contrast, the percentage of mutant CD8+ SP cells remained remarkably constant over the entire study (i.e., not reproducing WT characteristics) (Fig. 3B). Until ∼3–4 wk of age, only a small percentage of the WT CD8+ SP cells are of the mature (CD24lo) phenotype (Fig. 3C). Shortly thereafter, these cells seem to accumulate in thymus, reaching a plateau ∼6–10 wk after birth, before their fraction starts to gradually decline again. In contrast, the separate analysis of CD24lo versus CD24int/hiCD8+ mutant SP cells revealed that, by and large, the percentage of mature CD8+ SP cells remained at a low level, because the age-dependent accumulation of these cells does not take place when CD155 is missing (Fig. 3C).

FIGURE 3.

The CD155 phenotype develops only with time in BALB/c mice. The frequencies of SP cells were determined by flow cytometry from thymi of various ages. A, CD4+ SP thymocytes. B, CD8+ SP thymocytes. C, The CD8+ SP pool was separated into CD24lo and CD24int/hi fractions. The number of thymi analyzed (n) is shown below each bar. *p < 0.05; ***p < 0.001.

FIGURE 3.

The CD155 phenotype develops only with time in BALB/c mice. The frequencies of SP cells were determined by flow cytometry from thymi of various ages. A, CD4+ SP thymocytes. B, CD8+ SP thymocytes. C, The CD8+ SP pool was separated into CD24lo and CD24int/hi fractions. The number of thymi analyzed (n) is shown below each bar. *p < 0.05; ***p < 0.001.

Close modal

The selective absence of mature CD8+ SP cells in mutant thymi of BALB/c mice may be caused by a defective maturation, a premature exit, or an increased apoptotic rate of these particular cells. Determination of the latter did not reveal a significant difference between WT and mutant thymocytes (Fig. 4), even though apoptotic rates seemed to be slightly elevated in the KO mice.

FIGURE 4.

Apoptosis is not increased in CD155-deficient CD8+ SP thymocytes. Apoptotic CD8+ cells were identified by flow cytometry as Annexin-V+PI cells. Shown are representative scatter plots. Percentages ± SD result from the analysis of four WT and four CD155-deficient mice.

FIGURE 4.

Apoptosis is not increased in CD155-deficient CD8+ SP thymocytes. Apoptotic CD8+ cells were identified by flow cytometry as Annexin-V+PI cells. Shown are representative scatter plots. Percentages ± SD result from the analysis of four WT and four CD155-deficient mice.

Close modal

To address thymic maturation and the exit of mutant CD8 T cells, animals were treated for 10–12 d with FTY720, an immunomodulatory drug. FTY720 selectively interferes with S1P receptor activity, blocks the exit of mature T cells from thymus and pLNs, and induces phenotypic maturation of semimature SP thymocytes (18, 19, 42). Upon FTY720 application, the CD8+ SP cell frequency became indistinguishable between WT and mutant thymi, whereas in both cases, the overall number of SP cells increased considerably (Fig. 5A). This is most likely owed to the exit block triggered by the presence of FTY720. Subsequently, the profiles of expression of CD24, CD44, CD69, and CD62L were determined by flow cytometry (Fig. 5B and data not shown). The vast majority of SP cells in FTY720-treated thymi represent mature CD69loCD24loCD62hi cells. Most strikingly, WT and mutant cells gave rise to identical expression patterns of these maturation markers. In conjunction with the identical frequencies of CD8+ SP cells mentioned above, this indicates that CD155-deficient semimature SP thymocytes are able to mature and accumulate to an extent comparable to WT thymocytes. This suggests that the rates of SP cell production are similar, if not identical, in CD155-deficient thymi.

FIGURE 5.

FTY720 treatment abolished the differences in CD8+ SP composition caused by the lack of CD155. A, WT and CD155-deficient mice were fed FTY720 or PBSd as described in 1Materials and Methods. The thymi were analyzed for their content of SP cells 10–12 d later. Each circle represents one thymus. ***p < 0.001. B, SP cells were further characterized with regard to their expression of CD24, CD69, and CD62L. Representative graphs are shown.

FIGURE 5.

FTY720 treatment abolished the differences in CD8+ SP composition caused by the lack of CD155. A, WT and CD155-deficient mice were fed FTY720 or PBSd as described in 1Materials and Methods. The thymi were analyzed for their content of SP cells 10–12 d later. Each circle represents one thymus. ***p < 0.001. B, SP cells were further characterized with regard to their expression of CD24, CD69, and CD62L. Representative graphs are shown.

Close modal

We then analyzed the frequency of recent thymic emigrants (RTEs) by injecting the fluorescent dye FITC into WT and KO thymi. The dye unspecifically labels thymocytes that can be tracked as RTEs 40 h later when analyzing secondary lymphoid organs. For unknown reasons, the frequency of labeled mutant thymocytes was slightly less pronounced compared with WT thymocytes (Fig. 6A), yet we failed to note significant differences in the rates of RTEs in all secondary lymphoid organs analyzed. Although the numbers of RTEs are identical, the maturation stage may differ between WT and CD155-deficient RTEs. To test whether the latter are more or less mature compared with their WT counterparts, we analyzed the expression of CD44, CD62L, and CD24 on both types of RTEs. We observed that the levels of CD62L did not differ between RTEs and FITC peripheral CD8+ T cells, whereas RTEs had not yet completely downregulated CD44 and, in particular, CD24 to the levels found on the peripheral cells (Fig. 6B). This is in accordance with data published by other investigators (43, 44). More importantly, RTEs of CD155−/− and WT origin displayed an identical profile with regard to the expression of CD44, CD62L, and CD24. We conclude that the rate of production of mature SP thymocytes, as well as their rate of emigration, is not affected by CD155 deficiency.

FIGURE 6.

RTEs do not differ in frequency and phenotype between WT and CD155-deficient cells. A, FITC dye was injected intrathymically as described in 1Materials and Methods. The thymi, pLNs, and mLNs were analyzed 42 h later for CD8+ RTEs identified as FITC+ cells among unlabeled T cells. Each circle represents one animal. Closed circles: WT mice; open circles: CD155-deficient animals. B, Representative graphs show the expression pattern of CD62L, CD24, and CD44 in WT and CD155 deficient-CD8+ RTEs. The corresponding expression profile of peripheral naive CD8+ T cells that are FITC is also shown.

FIGURE 6.

RTEs do not differ in frequency and phenotype between WT and CD155-deficient cells. A, FITC dye was injected intrathymically as described in 1Materials and Methods. The thymi, pLNs, and mLNs were analyzed 42 h later for CD8+ RTEs identified as FITC+ cells among unlabeled T cells. Each circle represents one animal. Closed circles: WT mice; open circles: CD155-deficient animals. B, Representative graphs show the expression pattern of CD62L, CD24, and CD44 in WT and CD155 deficient-CD8+ RTEs. The corresponding expression profile of peripheral naive CD8+ T cells that are FITC is also shown.

Close modal

Taking into account that mutant CD8+ SP thymocytes did not differ from their WT counterparts, with regard to the critical parameters of emigration, maturation, and sensitivity to apoptosis, the most likely explanation for the observed paucity of mature CD8+ SP cells in mutant thymus is a defect in the capacity to retain these cells in the medulla during their final maturation. This is in line with the finding that not only SP thymocytes themselves express CD155 and its ligands CD226 and CD96 [(37, 40) and data not shown]. In addition, mTECs and thymic DCs are positive for CD155 (data not shown), thus providing a dense CD155-driven adhesive network in the medulla.

The finding that CD4+ SP cells and semimature CD8+ SP cells are not affected in mutant thymi suggests that positive selection proceeded normally in CD155-deficient cells. In general, positive selection determining lineage fate is assumed to precede negative selection. Passing through their final differentiation steps, thymocytes transit from the cortex to the medulla with semimature CD4+ SP cells commencing negative selection (2, 6). However, doubts were raised regarding the general validity of this selection schedule (45). In addition, virtually nothing is known about at which stage(s) negative selection occurs in the CD8 lineage. Provided that CD155 is involved in establishing or maintaining contacts with medullary cells, this may influence the dwell time of CD8+ SP cells as well as indirectly influence TCR-driven selection by decreasing the average TCR-mediated signal intensity (possibly also in the case of CD4+ SP cells). Therefore, it was of interest to investigate whether CD155 deficiency correlates with a shift in the TCR repertoire present in the pool of mutant SP cells. To this end, the expression rate of several TCR Vβ chains (Vβ2, 3, 4, 5, 6, 7, 8, 11, and 12) was determined by flow cytometry in 8–10-wk-old mice (Fig. 7A). The panel of chains analyzed was also guided by the earlier finding that a profound alteration in the frequency of diverse Vβ subsets especially prone to recognizing endogenous retroviral Ags is indicative of defective negative selection in the CD4+ cell pool. In adult BALB/c animals, the strain-specific retroviral Ags cause massive deletion of CD4+ T cells expressing Vβ3, 5, 11, and 12 (8, 9). When comparing the TCR Vβ repertoire between WT and mutant thymocytes, we observed moderate, but highly statistically significant, differences in the frequencies of Vβ2, 3, 4, 5, and 12 present on CD8+ SP cells, whereas only a marginal difference in Vβ8 usage was found for CD4+ SP cells. Likewise, the subchain composition in the DP fraction was virtually identical, suggesting that, up to this stage, T cell development is regular in CD155-deficient mice. These findings correlate well with the above-described selective deficiency in CD8+ SP cells, whereas CD4+ SP cells are largely unaffected; this supports the idea that adhesion receptor-mediated dwell time and negative selection represent coupled phenomena.

FIGURE 7.

Negative selection of CD8+ SP cells in WT and CD155-deficient mice. A, A series of Vβ chain-specific mAbs was exploited to determine the repertoire of TCRs expressed by WT and CD155-mutant DP cells, CD4+ SP cells, and CD8+ SP cells. Each circle represents one thymus. B, Separately analyzed Vβ repertoire in thymocytes of WT animals according to their CD24 expression. This was compared with the corresponding usage found in the CD8+CD62L+ fraction of mLN cells. A total of eight mice were analyzed in two separate experiments. Statistical analysis refers to the comparison of the CD24low fraction with the CD24int or mLN fraction. *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 7.

Negative selection of CD8+ SP cells in WT and CD155-deficient mice. A, A series of Vβ chain-specific mAbs was exploited to determine the repertoire of TCRs expressed by WT and CD155-mutant DP cells, CD4+ SP cells, and CD8+ SP cells. Each circle represents one thymus. B, Separately analyzed Vβ repertoire in thymocytes of WT animals according to their CD24 expression. This was compared with the corresponding usage found in the CD8+CD62L+ fraction of mLN cells. A total of eight mice were analyzed in two separate experiments. Statistical analysis refers to the comparison of the CD24low fraction with the CD24int or mLN fraction. *p < 0.05; **p < 0.01; ***p < 0.001.

Close modal

Surprisingly, a different result was obtained when analyzing peripheral naive T cells (Supplemental Fig. 1). Some of the observed shifts displayed by the mutant thymocytes appear attenuated in the periphery. CD8+ T cells isolated from mesenteric lymph nodes (mLNs) resemble their thymic counterparts with respect to Vβ4 but not Vβ2, 3, and 12 (no difference). Interestingly, the increased presence of Vβ5 on CD8+ SP thymocytes is opposed by a reduced frequency of this chain among peripheral CD8+ T cells.

In theory, the biases in the Vβ repertoire selection observed in the CD155-deficient CD8+ SP pool may represent a numerical artifact caused by the highly divergent contributions of the semimature CD24intCD8+ and mature CD24loCD8+ SP cell numbers in the KO scenario compared with the WT one. Such an assumption anticipates that the TCR Vβ distributions among semimature and mature CD8+ SP cells differ from each other, consequently postulating that negative selection operates predominantly in the semimature or mature stage. Following the concept that semimature CD24intCD8+ SP cells are sensitive to negative selection, as suggested for the CD4 lineage (6), it would be more appropriate to compare the CD24int fractions when determining whether negative selection is impaired in thymus of CD155-deficient mice. Likewise, the repertoire of the peripheral CD8+ T cells should be compared with that of the mature CD24loCD8+ thymocytes. Therefore, we analyzed the Vβ chain repertoire of semimature CD24intCD8+ and mature CD24loCD8+ SP cells in WT animals separately. Unfortunately, a corresponding investigation in CD155-deficient thymi was affected by the paucity of mature cells, rendering such an analysis statistically insignificant for low-frequency Vβ chains in the mature compartment. The results obtained from WT cells sustain the supposition of ongoing selection in the semimature CD24intCD8+ SP stage, because statistically significant differences in the usage of Vβ chains 3, 4, 5, 6, 7, and 11 were found compared with mature CD8+ SP cells (Fig. 7B). In support of this, the frequency patterns of CD24loCD8+ SP cells and peripheral naive CD8+ T cells resemble each other more closely than do those of CD24loCD8+ and CD24intCD8+ SP thymocytes. This is in accordance with the above-mentioned model. Nevertheless, the minor divergences in Vβ usage found between the mature CD24loCD8+ thymocytes and peripheral CD8+ T cells (Vβ2, 6, 7, 8, 11, and 12) indicate an ongoing shaping of the TCR repertoire finally engaged by peripheral naive CD8+ T cells. However, it is uncertain whether such fine-tuning occurs in the pool of mature thymocytes and/or in the periphery.

When comparing the WT CD24int fraction with the CD155-deficient CD8+ SP population that is dominated by the CD24int pool, it is evident that the original differences in Vβ3, 5, and 12 frequency found were largely caused by the biased contributions of the semimature and mature cells to the pool of mutant CD8+ SP cells (Supplemental Fig. 2). However, this does not apply for Vβ2, and particularly not for Vβ4. These discrepancies in the repertoire selection between WT and KO CD8+ thymocytes must have come into existence before cells completed their transition to the mature stage.

This assumption is supported by another observation. As shown above, following treatment with FTY720, the composition of the CD8 SP compartment no longer differed between WT and CD155-deficient thymi (Fig. 5). When analyzing the frequencies of Vβ2-, 3-, 4-, 5-, and 12-expressing T cells, we observed that FTY720 application equalized the different frequencies in Vβ3, 5, and 12 but not in Vβ2 and 4 (Supplemental Fig. 3). Remarkably, however, Vβ4 was slightly increased by FTY720 treatment in KO animals, whereas it was reduced in untreated mice compared with the corresponding WT controls. Such a phenomenon may relate to the finding that FTY720 can interfere with negative selection (46).

Considering this, the shortened residency of mature CD8+ SP cells caused by CD155 deficiency and the concomitant bias in the repertoire selection may not always share a common origin (i.e., negative selection and length of stay do not trace back to the same adhesive events); thus, they may be mediated by different cell types contacted by the thymocytes. However, a defect in adhesion may account for both phenomena, and both seem to overlap chronologically; the results presented above admit the interpretation that negative selection continues to shape the TCR repertoire to a certain extent beyond the semimature stage. Thus, a prolonged residency of CD8+ SP cells in medulla beginning with adolescence may influence the TCR repertoire of naive CD8+ cells by increasing the probability of productive encounters between selectors and CD8+ thymocytes.

When analyzing thymi of CD155-deficient mice, we found a significant reduction in the frequency of CD8+ SP but not CD4+ SP cells. Inclusion of maturation markers, such as CD24, CD69, or CD62L, in the flow cytometric analyses allowed assignment of the observed phenotype to the most mature CD8 cell population present in thymus: CD24loCD69loCD62Lhi cells ready to exit into the periphery. A further refinement of the investigations of this cell type including thymi of various ages revealed that the frequency profile of mature WT CD8+ SP cells is subject to significant variations, whereas that of semimature CD8+ SP cells remained remarkably constant. Beginning at ∼3–4 wk after birth, mature CD8+ SP cells accumulate in thymus, reaching a plateau at 6–10 wk. In contrast, the frequencies of mature CD8+ SP cells remained invariantly low in CD155-deficient mice of all ages analyzed. Regarding these dynamics, the BALB/c CD155−/− thymocytes displayed characteristics resembling mature WT C57BL/6 CD8+ SP cells that failed to accumulate during adolescence (Fig. 2 and data not shown). Interestingly, the genetic background seems to exert a profound influence on the capability of mature CD8+ SP cells to accumulate in thymus, but conclusive statements await more detailed analyses for various strains as done in this study for BALB/c animals. A strain-dependent modulation of SP frequencies and population dynamics (potentially also affecting CD4+ SP cells) represents a thus-far neglected parameter in the discussion of experimental results addressing, for example, the dwell time of SP cells in thymus (see Ref. 47 for a concise overview). Therefore, considering this aspect may help to reconcile conflicting interpretations of studies that used different mouse model strains.

CD155 represents an adhesion receptor. Together with the results obtained from other experiments presented in this study (FITC injection, FTY720 treatment, and determination of apoptosis rate), we hypothesize that matured CD8+ SP cells cannot be retained in the medulla of BALB/c thymi under CD155 deficiency, thereby curtailing their dwell time in this compartment. In general, the length of time that CD8+ SP cells spend in the medulla may correlate positively with the efficacy/duration and the frequency of adhesion events that occur between themselves and other developing thymocytes, stroma cells, DCs, or extracellular matrix. Currently, we can provide only provisional evidence that the lack of mature CD8+ SP cells is caused primarily by a thymocyte-born defect or by CD155 deficiency on DCs. The analyses of bone marrow chimeric animals were obscured by the severe side effects of irradiation; it caused a substantial loss of mature CD8+ SP cells in the control group, which consisted of WT animals that had received WT bone marrow (Supplemental Fig. 4). In this study, we observed a much lower frequency of CD8+ SP cells and a ratio of semimature/mature CD8+ SP cells of 5.66 ± 0.43, which resembled that found in untreated BALB/c KO or BL6 mice (Fig. 2C). Yet we noticed that the residual retention of the mature cells in irradiated mice was due to the presence of CD155 on T cells and/or DCs, because KO animals reconstituted with WT bone marrow displayed a similar ratio of semimature/mature CD8+ SP cells (4.69 ± 1.30). In contrast, in KO to WT reconstituted animals, an even more pronounced deficiency of mature CD8+ SP cells (ratio, 8.34 ± 0.70) was found, which approached the ratio seen in the KO/KO control group (11.19 ± 1.54).

By separately analyzing the TCR repertoire present on semimature, mature CD8+ SP cells and naive peripheral CD8+ T cells, it was possible to document an ongoing selection process in otherwise nonmanipulated mice. Even if we studied only a selected set of Vβ chains, the comparison of their patterns would suggest that the main step in repertoire shaping is completed in the stage of semimature CD24int CD8+ SP cells. This is illustrated by the finding that mature CD8+ SP thymocytes and their peripheral counterparts diverged only modestly from each other with regard to their TCR repertoire. We provided evidence that CD155 participates in delaying the exit of mature CD8+ SP thymocytes and influences TCR repertoire selection. As outlined above, these two phenotypes may be evoked by the same type of molecular adhesion events but in the context of different cellular interaction partners. Whatever applies, the premature release of cells distinguished by uncompleted thymic selection may cause problems in that potentially autoreactive cells escape into the periphery. However, such cells may be kept in check by regulatory T cells as well as by an operative peripheral negative selection in a fashion resembling thymic self-education including Aire-expressing cells (48). Particularly, the inverted frequencies of the Vβ5 chain expressed by mature thymic versus peripheral CD8+ T cells would support such an assumption. Thus, there is evidence that the TCR repertoire is continuously surveyed and shaped at many stages inside thymus as well as in the periphery. The Ags required for presentation are made available locally or by transporting them into thymus as well as by expressing them in the periphery on DCs or in a manner comparable to mTECs (6, 4851). However, a drift in the TCR repertoire in the pool of peripheral naive CD8+ T cells may also be evoked by a biased self-renewal or a preferential supply of survival signals to cells displaying distinct TCRs. The interpretation of the results is complicated further by the concept that thymocyte development may follow slightly different guidelines in adults compared with juvenile (<3–4-wk-old) mice; this may also affect the selective forces driving the adjustment of the TCR repertoire in use. Such an idea is supported by the fact that we failed to find significant differences when comparing WT and KO CD8+ SP cells isolated from 3–4-wk-old mice (with the exception of Vβ12 being slightly underrepresented on CD155-deficient cells [Supplemental Fig. 5]). Moreover, the juvenile Vβ repertoire is significantly different from the corresponding composition in adulthood. Thus, the output of RTEs differing in their TCR signature from previous waves may slowly, but constantly, supersede the repertoire currently in use in the periphery (52).

It is remarkable that CD155 deficiency almost exclusively affects adult CD8+ SP cells. Our observations illustrate that the developing thymocytes rely on different adhesion systems that depend on the T cell subset as well as their developmental stage (semimature versus mature). Moreover, starting at ∼3–4 wk, a change in the adhesive settings causes an accumulation of mature CD8+ SP cells in thymus now critically engaging CD155. This event may correlate with birth. It is estimated that one round of thymocyte development requires ∼3 wk after the precursors start their differentiation program (2). Filling of thymus with precursors occurs in waves (53), and it seems that the first wave of prothymocytes settling the organ perinatally follows a slightly different path of maturation. This fits into a time frame that the majority of freshly produced mature T cells 3–4 wk later consists of progeny derived from the perinatal immigration wave. Interestingly, at the same time, animals experience some profound immunological changes related to weaning. A reorientation of the immune system is required to accommodate this change in the individual’s exposure to exogenous burden regarding harmless Ags, such as food, as well as commensal microflora and pathogens. The end of weaning and the concomitant discontinuation of the protective maternal Ig shield coincide with structural alterations/maturation in GALT and remote lymphoid organs, a phenomenon that was found to correlate with commensal microflora now present in the gut (5456). As outlined above, newly released RTEs refresh the precedent peripheral pool of T cells, possibly facilitating to adopt to the requirement of changing immunological challenges met during subsequent stages in life (52). Therefore, it is tempting to speculate that the generation of CD8+ T cells starting to settle the periphery at ∼3–4 wk must fulfill different criteria that manifest as an extended residency in the medulla.

Taken together, the discrepancies in repertoire selection as well as age-dependent dwell time of CD8+ SP thymocytes, but not CD4+ SP thymocytes, related to CD155 function helped disclose hidden aspects of the life cycle of terminally maturing CD8+ SP cells. To our knowledge, the provoked abnormalities due to CD155 deficiency are not sufficient to elicit macroscopic phenotypes, such as overt signs of autoimmunity, even in old mice (data not shown). However, final conclusions regarding these aspects await more detailed analyses of CD155-deficient mice.

We thank O. Pabst for critically reading the manuscript.

Disclosures The authors have no financial conflicts of interest.

This work was supported by Grants BE1886/2-1/-2 from the Deutsche Forschungsgemeinschaft. Q.Q. was supported by the Wilhelm Hirte Foundation. S.S. and A.R. were awarded a Georg Christoph Lichtenberg scholarship provided by the Ph.D. Program in Infection Biology sponsored by the Ministry of Science and Culture of Lower Saxony.

The online version of this article contains supplemental material.

Abbreviations used in this paper:

DC

dendritic cell

DP

double-positive

ISP

immature single-positive

KO

knockout

mLN

mesenteric lymph node

mTEC

medullary epithelial cell

pLN

peripheral lymph node

RTE

recent thymic emigrant

S1P

sphingosine 1-phosphate

SP

single-positive

WT

wild-type.

1
Hayday
A. C.
,
Pennington
D. J.
.
2007
.
Key factors in the organized chaos of early T cell development.
Nat. Immunol.
8
:
137
144
.
2
Mißlitz
A.
,
Bernhardt
G.
,
Förster
R.
.
2006
.
Trafficking on serpentines: molecular insight on how maturating T cells find their winding paths in the thymus.
Immunol. Rev.
209
:
115
128
.
3
Singer
A.
,
Adoro
S.
,
Park
J. H.
.
2008
.
Lineage fate and intense debate: myths, models and mechanisms of CD4- versus CD8-lineage choice.
Nat. Rev. Immunol.
8
:
788
801
.
4
McCaughtry
T. M.
,
Baldwin
T. A.
,
Wilken
M. S.
,
Hogquist
K. A.
.
2008
.
Clonal deletion of thymocytes can occur in the cortex with no involvement of the medulla.
J. Exp. Med.
205
:
2575
2584
.
5
Hogquist
K. A.
,
Baldwin
T. A.
,
Jameson
S. C.
.
2005
.
Central tolerance: learning self-control in the thymus.
Nat. Rev. Immunol.
5
:
772
782
.
6
Sprent
J.
,
Kishimoto
H.
.
2002
.
The thymus and negative selection.
Immunol. Rev.
185
:
126
135
.
7
Anderson
M. S.
,
Venanzi
E. S.
,
Chen
Z.
,
Berzins
S. P.
,
Benoist
C.
,
Mathis
D.
.
2005
.
The cellular mechanism of Aire control of T cell tolerance.
Immunity
23
:
227
239
.
8
Davalos-Misslitz
A. C.
,
Worbs
T.
,
Willenzon
S.
,
Bernhardt
G.
,
Förster
R.
.
2007
.
Impaired responsiveness to T-cell receptor stimulation and defective negative selection of thymocytes in CCR7-deficient mice.
Blood
110
:
4351
4359
.
9
Foy
T. M.
,
Page
D. M.
,
Waldschmidt
T. J.
,
Schoneveld
A.
,
Laman
J. D.
,
Masters
S. R.
,
Tygrett
L.
,
Ledbetter
J. A.
,
Aruffo
A.
,
Claassen
E.
, et al
.
1995
.
An essential role for gp39, the ligand for CD40, in thymic selection.
J. Exp. Med.
182
:
1377
1388
.
10
Kishimoto
H.
,
Sprent
J.
.
1999
.
Several different cell surface molecules control negative selection of medullary thymocytes.
J. Exp. Med.
190
:
65
73
.
11
Takahashi
S.
,
Kataoka
H.
,
Hara
S.
,
Yokosuka
T.
,
Takase
K.
,
Yamasaki
S.
,
Kobayashi
W.
,
Saito
Y.
,
Saito
T.
.
2005
.
In vivo overexpression of CTLA-4 suppresses lymphoproliferative diseases and thymic negative selection.
Eur. J. Immunol.
35
:
399
407
.
12
Schmeissner
P. J.
,
Xie
H.
,
Smilenov
L. B.
,
Shu
F.
,
Marcantonio
E. E.
.
2001
.
Integrin functions play a key role in the differentiation of thymocytes in vivo.
J. Immunol.
167
:
3715
3724
.
13
Kutlesa
S.
,
Wessels
J. T.
,
Speiser
A.
,
Steiert
I.
,
Müller
C. A.
,
Klein
G.
.
2002
.
E-cadherin-mediated interactions of thymic epithelial cells with CD103+ thymocytes lead to enhanced thymocyte cell proliferation.
J. Cell Sci.
115
:
4505
4515
.
14
Revilla
C.
,
González
A. L.
,
Conde
C.
,
López-Hoyos
M.
,
Merino
J.
.
1997
.
Treatment with anti-LFA-1 alpha monoclonal antibody selectively interferes with the maturation of CD4− 8+ thymocytes.
Immunology
90
:
550
556
.
15
Ge
Q.
,
Chen
W. F.
.
1999
.
Phenotypic identification of the subgroups of murine T-cell receptor alphabeta+ CD4+ CD8− thymocytes and its implication in the late stage of thymocyte development.
Immunology
97
:
665
671
.
16
Kishimoto
H.
,
Sprent
J.
.
1997
.
Negative selection in the thymus includes semimature T cells.
J. Exp. Med.
185
:
263
271
.
17
Weinreich
M. A.
,
Hogquist
K. A.
.
2008
.
Thymic emigration: when and how T cells leave home.
J. Immunol.
181
:
2265
2270
.
18
Rosen
H.
,
Alfonso
C.
,
Surh
C. D.
,
McHeyzer-Williams
M. G.
.
2003
.
Rapid induction of medullary thymocyte phenotypic maturation and egress inhibition by nanomolar sphingosine 1-phosphate receptor agonist.
Proc. Natl. Acad. Sci. USA
100
:
10907
10912
.
19
Matloubian
M.
,
Lo
C. G.
,
Cinamon
G.
,
Lesneski
M. J.
,
Xu
Y.
,
Brinkmann
V.
,
Allende
M. L.
,
Proia
R. L.
,
Cyster
J. G.
.
2004
.
Lymphocyte egress from thymus and peripheral lymphoid organs is dependent on S1P receptor 1.
Nature
427
:
355
360
.
20
Mendelsohn
C. L.
,
Wimmer
E.
,
Racaniello
V. R.
.
1989
.
Cellular receptor for poliovirus: molecular cloning, nucleotide sequence, and expression of a new member of the immunoglobulin superfamily.
Cell
56
:
855
865
.
21
Takai
Y.
,
Irie
K.
,
Shimizu
K.
,
Sakisaka
T.
,
Ikeda
W.
.
2003
.
Nectins and nectin-like molecules: roles in cell adhesion, migration, and polarization.
Cancer Sci.
94
:
655
667
.
22
Bernhardt
G.
,
Harber
J. J.
,
Zibert
A.
,
deCrombrugghe
M.
,
Wimmer
E.
.
1994
.
The poliovirus receptor: identification of domains and amino acid residues critical for virus binding.
Virology
203
:
344
356
.
23
Bottino
C.
,
Castriconi
R.
,
Pende
D.
,
Rivera
P.
,
Nanni
M.
,
Carnemolla
B.
,
Cantoni
C.
,
Grassi
J.
,
Marcenaro
S.
,
Reymond
N.
, et al
.
2003
.
Identification of PVR (CD155) and Nectin-2 (CD112) as cell surface ligands for the human DNAM-1 (CD226) activating molecule.
J. Exp. Med.
198
:
557
567
.
24
Fuchs
A.
,
Cella
M.
,
Giurisato
E.
,
Shaw
A. S.
,
Colonna
M.
.
2004
.
Cutting edge: CD96 (tactile) promotes NK cell-target cell adhesion by interacting with the poliovirus receptor (CD155).
J. Immunol.
172
:
3994
3998
.
25
Ikeda
W.
,
Kakunaga
S.
,
Itoh
S.
,
Shingai
T.
,
Takekuni
K.
,
Satoh
K.
,
Inoue
Y.
,
Hamaguchi
A.
,
Morimoto
K.
,
Takeuchi
M.
, et al
.
2003
.
Tage4/Nectin-like molecule-5 heterophilically trans-interacts with cell adhesion molecule Nectin-3 and enhances cell migration.
J. Biol. Chem.
278
:
28167
28172
.
26
Lange
R.
,
Peng
X.
,
Wimmer
E.
,
Lipp
M.
,
Bernhardt
G.
.
2001
.
The poliovirus receptor CD155 mediates cell-to-matrix contacts by specifically binding to vitronectin.
Virology
285
:
218
227
.
27
Meyer
D.
,
Seth
S.
,
Albrecht
J.
,
Maier
M. K.
,
du Pasquier
L.
,
Ravens
I.
,
Dreyer
L.
,
Burger
R.
,
Gramatzki
M.
,
Schwinzer
R.
, et al
.
2009
.
CD96 interaction with CD155 via its first Ig-like domain is modulated by alternative splicing or mutations in distal Ig-like domains.
J. Biol. Chem.
284
:
2235
2244
.
28
Mueller
S.
,
Wimmer
E.
.
2003
.
Recruitment of nectin-3 to cell-cell junctions through trans-heterophilic interaction with CD155, a vitronectin and poliovirus receptor that localizes to alpha(v)beta3 integrin-containing membrane microdomains.
J. Biol. Chem.
278
:
31251
31260
.
29
Ravens
I.
,
Seth
S.
,
Förster
R.
,
Bernhardt
G.
.
2003
.
Characterization and identification of Tage4 as the murine orthologue of human poliovirus receptor/CD155.
Biochem. Biophys. Res. Commun.
312
:
1364
1371
.
30
Ogita
H.
,
Takai
Y.
.
2006
.
Nectins and nectin-like molecules: roles in cell adhesion, polarization, movement, and proliferation.
IUBMB Life
58
:
334
343
.
31
Sakamoto
Y.
,
Ogita
H.
,
Komura
H.
,
Takai
Y.
.
2008
.
Involvement of nectin in inactivation of integrin alpha(v)beta(3) after the establishment of cell-cell adhesion.
J. Biol. Chem.
283
:
496
505
.
32
Castriconi
R.
,
Dondero
A.
,
Corrias
M. V.
,
Lanino
E.
,
Pende
D.
,
Moretta
L.
,
Bottino
C.
,
Moretta
A.
.
2004
.
Natural killer cell-mediated killing of freshly isolated neuroblastoma cells: critical role of DNAX accessory molecule-1-poliovirus receptor interaction.
Cancer Res.
64
:
9180
9184
.
33
Gilfillan
S.
,
Chan
C. J.
,
Cella
M.
,
Haynes
N. M.
,
Rapaport
A. S.
,
Boles
K. S.
,
Andrews
D. M.
,
Smyth
M. J.
,
Colonna
M.
.
2008
.
DNAM-1 promotes activation of cytotoxic lymphocytes by nonprofessional antigen-presenting cells and tumors.
J. Exp. Med.
205
:
2965
2973
.
34
Iguchi-Manaka
A.
,
Kai
H.
,
Yamashita
Y.
,
Shibata
K.
,
Tahara-Hanaoka
S.
,
Honda
S.
,
Yasui
T.
,
Kikutani
H.
,
Shibuya
K.
,
Shibuya
A.
.
2008
.
Accelerated tumor growth in mice deficient in DNAM-1 receptor.
J. Exp. Med.
205
:
2959
2964
.
35
Pende
D.
,
Castriconi
R.
,
Romagnani
P.
,
Spaggiari
G. M.
,
Marcenaro
S.
,
Dondero
A.
,
Lazzeri
E.
,
Lasagni
L.
,
Martini
S.
,
Rivera
P.
, et al
.
2006
.
Expression of the DNAM-1 ligands, Nectin-2 (CD112) and poliovirus receptor (CD155), on dendritic cells: relevance for natural killer-dendritic cell interaction.
Blood
107
:
2030
2036
.
36
Seth
S.
,
Georgoudaki
A. M.
,
Chambers
B. J.
,
Qiu
Q.
,
Kremmer
E.
,
Maier
M. K.
,
Czeloth
N.
,
Ravens
I.
,
Foerster
R.
,
Bernhardt
G.
.
2009
.
Heterogeneous expression of the adhesion receptor CD226 on murine NK and T cells and its function in NK-mediated killing of immature dendritic cells.
J. Leukoc. Biol.
86
:
91
101
.
37
Maier
M. K.
,
Seth
S.
,
Czeloth
N.
,
Qiu
Q.
,
Ravens
I.
,
Kremmer
E.
,
Ebel
M.
,
Müller
W.
,
Pabst
O.
,
Förster
R.
,
Bernhardt
G.
.
2007
.
The adhesion receptor CD155 determines the magnitude of humoral immune responses against orally ingested antigens.
Eur. J. Immunol.
37
:
2214
2225
.
38
Seth
S.
,
Ravens
I.
,
Kremmer
E.
,
Maier
M. K.
,
Hadis
U.
,
Hardtke
S.
,
Förster
R.
,
Bernhardt
G.
.
2009
.
Abundance of follicular helper T cells in Peyer’s patches is modulated by CD155.
Eur. J. Immunol.
39
:
3160
3170
.
39
Czeloth
N.
,
Bernhardt
G.
,
Hofmann
F.
,
Genth
H.
,
Förster
R.
.
2005
.
Sphingosine-1-phosphate mediates migration of mature dendritic cells.
J. Immunol.
175
:
2960
2967
.
40
Seth
S.
,
Maier
M. K.
,
Qiu
Q.
,
Ravens
I.
,
Kremmer
E.
,
Förster
R.
,
Bernhardt
G.
.
2007
.
The murine pan T cell marker CD96 is an adhesion receptor for CD155 and nectin-1.
Biochem. Biophys. Res. Commun.
364
:
959
965
.
41
Penit
C.
,
Vasseur
F.
.
1989
.
Cell proliferation and differentiation in the fetal and early postnatal mouse thymus.
J. Immunol.
142
:
3369
3377
.
42
Yagi
H.
,
Kamba
R.
,
Chiba
K.
,
Soga
H.
,
Yaguchi
K.
,
Nakamura
M.
,
Itoh
T.
.
2000
.
Immunosuppressant FTY720 inhibits thymocyte emigration.
Eur. J. Immunol.
30
:
1435
1444
.
43
Boursalian
T. E.
,
Golob
J.
,
Soper
D. M.
,
Cooper
C. J.
,
Fink
P. J.
.
2004
.
Continued maturation of thymic emigrants in the periphery.
Nat. Immunol.
5
:
418
425
.
44
Gabor
M. J.
,
Godfrey
D. I.
,
Scollay
R.
.
1997
.
Recent thymic emigrants are distinct from most medullary thymocytes.
Eur. J. Immunol.
27
:
2010
2015
.
45
von Boehmer
H.
,
Kisielow
P.
.
2006
.
Negative selection of the T-cell repertoire: where and when does it occur?
Immunol. Rev.
209
:
284
289
.
46
Shimizu
C.
,
Li
X.
,
Kimura
M.
,
Hashimoto
K.
,
Sugaya
K.
,
Kubo
M.
,
Suzuki
S.
,
Nakayama
T.
.
1998
.
A novel immunosuppressant, FTY720, increases the efficiency of a superantigen-induced peripheral T-cell deletion whilst inhibiting negative selection in the thymus.
Immunology
94
:
503
512
.
47
McCaughtry
T. M.
,
Wilken
M. S.
,
Hogquist
K. A.
.
2007
.
Thymic emigration revisited.
J. Exp. Med.
204
:
2513
2520
.
48
Gardner
J. M.
,
Devoss
J. J.
,
Friedman
R. S.
,
Wong
D. J.
,
Tan
Y. X.
,
Zhou
X.
,
Johannes
K. P.
,
Su
M. A.
,
Chang
H. Y.
,
Krummel
M. F.
,
Anderson
M. S.
.
2008
.
Deletional tolerance mediated by extrathymic Aire-expressing cells.
Science
321
:
843
847
.
49
Steinman
R. M.
,
Hawiger
D.
,
Nussenzweig
M. C.
.
2003
.
Tolerogenic dendritic cells.
Annu. Rev. Immunol.
21
:
685
711
.
50
Probst
H. C.
,
McCoy
K.
,
Okazaki
T.
,
Honjo
T.
,
van den Broek
M.
.
2005
.
Resting dendritic cells induce peripheral CD8+ T cell tolerance through PD-1 and CTLA-4.
Nat. Immunol.
6
:
280
286
.
51
Bonasio
R.
,
Scimone
M. L.
,
Schaerli
P.
,
Grabie
N.
,
Lichtman
A. H.
,
von Andrian
U. H.
.
2006
.
Clonal deletion of thymocytes by circulating dendritic cells homing to the thymus.
Nat. Immunol.
7
:
1092
1100
.
52
Tanchot
C.
,
Rocha
B.
.
1997
.
Peripheral selection of T cell repertoires: the role of continuous thymus output.
J. Exp. Med.
186
:
1099
1106
.
53
Foss
D. L.
,
Donskoy
E.
,
Goldschneider
I.
.
2001
.
The importation of hematogenous precursors by the thymus is a gated phenomenon in normal adult mice.
J. Exp. Med.
193
:
365
374
.
54
Förster
R.
,
Pabst
O.
,
Bernhardt
G.
.
2008
.
Homeostatic chemokines in development, plasticity, and functional organization of the intestinal immune system.
Semin. Immunol.
20
:
171
180
.
55
Hooper
L. V.
2004
.
Bacterial contributions to mammalian gut development.
Trends Microbiol.
12
:
129
134
.
56
Mazmanian
S. K.
,
Liu
C. H.
,
Tzianabos
A. O.
,
Kasper
D. L.
.
2005
.
An immunomodulatory molecule of symbiotic bacteria directs maturation of the host immune system.
Cell
122
:
107
118
.