CD226 interaction with CD155 impacts on retention and negative selection of CD8 positive thymocytes as well as T cell differentiation to follicular helper cells in Peyer's Patches.

The immunoglobulin-like glycoprotein CD226 represents a receptor activating cytotoxic T and NK cells taking part in tumour surveillance. In addition, CD226 is involved in the differentiation of naïve CD4(+) T cells into effector cells. CD155 that is widely over-expressed on tumour cells, was identified as a counter-receptor of CD226 rendering many cancer cells sensitive to NK driven elimination. However, CD155 was also assigned a role in the establishment of follicular helper T cells in the small intestine and the final maturation of CD8 positive thymocytes. Here we show that mice lacking CD226 are distinguished by virtually identical phenotypes as already reported for CD155 deficient mice: a paucity of follicular helper T cells in Peyer's Patches and of terminally matured CD8 T cells in thymus. Moreover, like CD155, CD226 is involved in negative selection of CD8 thymocytes. These observations establish a firm link between the functions of CD155 and CD226 in several T cell differentiation steps.

Intranodal interaction with dendritic cells dynamically regulates surface expression of the co-stimulatory receptor CD226 protein on murine T cells.

Dendritic cells (DCs) are the most potent antigen-presenting cells of the immune system. Depending on their maturation status, they prime T cells to induce adaptive immunity or tolerance. DCs express CD155, an immunoglobulin-like receptor binding CD226 present on T and natural killer (NK) cells. CD226 represents an important co-stimulator during T cell priming but also serves as an activating receptor on cytotoxic T and NK cells. Here, we report that cells of the T and NK cell lineage of CD155(-/-) mice express markedly elevated protein levels of CD226 compared with wild type (WT). On heterozygous CD155(+/-) T cells, CD226 up-regulation is half-maximal, implying an inverse gene-dosis effect. Moreover, CD226 up-regulation is independent of antigen-driven activation because it occurs already in thymocytes and naïve peripheral T cells. In vivo, neutralizing anti-CD155 antibody elicits up-regulation of CD226 on T cells demonstrating, that the observed modulation can be triggered by interrupting CD155-CD226 contacts. Adoptive transfers of WT or CD155(-/-) T cells into CD155(-/-) or WT recipients, respectively, revealed that CD226 modulation is accomplished in trans. Analysis of bone marrow chimeras showed that regulators in trans are of hematopoietic origin. We demonstrate that DCs are capable of manipulating CD226 levels on T cells in vivo but not in vitro, suggesting that the process of T cells actively scanning antigen-presenting DCs inside secondary lymphoid organs is required for CD226 modulation. Hence, a CD226 level divergent from WT may be exploited as a sensor to detect abnormal DC/T-cell cross-talk as illustrated for T cells in mice lacking CCR7.

Tolerance induction towards cardiac allografts under costimulation blockade is impaired in CCR7-deficient animals but can be restored by adoptive transfer of syngeneic plasmacytoid dendritic cells.

Deficiency of transplant recipients for the chemokine receptor CCR7 was originally described to slightly increase the survival time of vascularized solid organ grafts, probably due to a reduced priming of alloreactive T cells. Using a model of allotolerance induction by donor-specific splenocyte transfusion (DST) in combination with anti-CD40L mAb-mediated costimulation blockade (CSB), we show here a striking failure of CCR7-deficient (CCR7(-/-) ) recipients to tolerate cardiac allografts. Furthermore, in addition to the recently described lack of Treg, CCR7(-/-) mice were found to harbor significantly reduced numbers of plasmacytoid dendritic cells (pDCs) within peripheral as well as mesenteric lymph nodes (LNs), but not the bone marrow or spleen. pDCs had previously been suggested to function as tolerogenic APC during allograft transplantation, and a single transfer of syngeneic WT pDCs, but not conventional DCs, was indeed sufficient to rescue graft survival in DST+CSB-treated CCR7(-/-) recipients in a dose-dependent manner. We therefore conclude that the nearly complete absence of pDCs within LNs of CCR7(-/-) mice prevents the successful induction of DST+CSB-mediated allotolerance, leading to the observed acute rejection of cardiac allografts under tolerizing conditions.

CCR7 essentially contributes to the homing of plasmacytoid dendritic cells to lymph nodes under steady-state as well as inflammatory conditions.

The chemokine receptor CCR7 represents an important determinant for circulating lymphocytes to enter lymph nodes (LN) via high endothelial venules. High endothelial venules also represent the major site of entry for plasmacytoid dendritic cells (pDC). In the steady-state, murine pDC have been suggested to home to LN engaging the chemokine receptors CXCR3, CXCR4, and CCR5, whereas responsiveness to CCR7 ligands is thought to be acquired only upon activation. In this study, we show that already resting pDC express minute amounts of CCR7 that suffice to trigger migration to CCL19/CCL21 in vitro. Upon activation with TLR ligands, CCR7 levels on pDC are strongly increased. Notably, CCR7-deficient mice display substantially reduced pDC counts in LN but not in bone marrow and spleen. Adoptive cell transfer experiments revealed that under both steady-state as well as inflammatory conditions, the homing of CCR7-deficient pDC is severely impaired, indicating that the reduced cell counts of naive pDC observed in CCR7(-/-) mice reflect an intrinsic homing defect of pDC. Together, these observations provide strong evidence that similar to naive lymphocytes, nonstimulated pDC exploit CCR7 to gain entry into LN. This adds to the repertoire of chemokine receptors permitting them to enter diverse tissues.

CD155 is involved in negative selection and is required to retain terminally maturing CD8 T cells in thymus.

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.

Antigen-dependent rescue of nose-associated lymphoid tissue (NALT) development independent of LTbetaR and CXCR5 signaling.

Nose-associated lymphoid tissue (NALT) in the rodent upper respiratory tract develops postnatally and is considered to be independent of several factors known to be involved in the organogenesis of LN and Peyer's patches (PP). In this study we demonstrate that at least two different pathways result in NALT development. Following NALT anlage formation the intrinsic pathway relies on a signaling cascade including those mediated through the chemokine receptor CXCR5 and the lymphotoxin beta receptor (LTbetaR). This allows for the formation of high endothelial venules and thereby the recruitment of lymphocytes into NALT. Alternatively, high endothelial venule formation and lymphocyte recruitment can be induced in the NALT anlage by environmental signals, which are independent of LT-betaR and chemokine receptor CXCR5 signaling but in part rely on CD40 ligand. Thus, our study identifies a novel mechanism that facilitates the rescue of NALT development at late stages in adult life independent of the canonical LTbetaR-CXCR5 signaling axis.

Abundance of follicular helper T cells in Peyer's patches is modulated by CD155.

The secondary humoral immune response is characterized by plasma B cells secreting isotype-switched and affinity-matured antibodies. The efficient generation of plasma B cells in the GC depends on the presence of follicular helper T (T(FH)) cells, a cell type thought to arise from naive CD4-positive T cells by a hitherto unresolved differentiation pathway. Mice deficient for CD155, an adhesion receptor of the immunoglobulin superfamily, are impaired to mount a secondary humoral immune response upon oral administration of antigen, while the primary IgM response is unaffected. Here, we show that mice lacking CD155 harbor significantly reduced numbers of T(FH) cells in their Peyer's patches. This was paralleled by a decreased frequency of T(FH) cells in the GC. Moreover, the CD155 ligand CD226, which is involved in T-cell activation, is down-regulated during T(FH) cell differentiation, resulting in a complete absence of CD226 on those T(FH) cells residing in the GC. Concurrently, the expression of TIGIT/WUCAM, a newly discovered CD155 ligand, is induced in T(FH) cells. Thus, these cells replace an activating by a putative inhibitory CD155-binding partner during their differentiation.

Heterogeneous expression of the adhesion receptor CD226 on murine NK and T cells and its function in NK-mediated killing of immature dendritic cells.

The adhesion receptor CD226 (DNAM-1) is a member of the Ig superfamily possessing two extracellular V-like domains. In humans, CD226 was shown to be expressed by NK as well as T cells. During T cell priming, CD226-mediated costimulatory signals may skew the subsequent differentiation into the Th1 pathway. In addition, CD226 expressed on NK and cytotoxic T cells is engaged by its counter-receptor CD155, present on target cells, thereby triggering their elimination. We established mAb specifically recognizing mCD226, demonstrating that CD226 is expressed by precursor and mature but not developing T cells. In contrast, NK cells are distinguished by a rather heterogeneous CD226 expression profile. In addition, expression of CD226 appears coupled to that of other NK cell receptors, as high expression of CD226 was found to correlate with decreased proportions of Ly49D and H positive NK cells. Upon injection into mice, the anti-CD226 antibodies caused selective depletion of CD8(+) T cells. Moreover, these antibodies as well as a naturally occurring CD226 splice variant lacking the outermost V-like domain were instrumental in determining that CD226 adheres to CD155 via its first domain. In addition, antibodies were identified as capable of blocking the CD226/CD155 interaction and to prevent NK-driven killing of immature DC. CD226 is thus the first mNK receptor identified to be essential for the elimination of this particular cell type.

CD96 interaction with CD155 via its first Ig-like domain is modulated by alternative splicing or mutations in distal Ig-like domains.

The adhesion receptor CD96 (TACTILE) is a transmembrane glycoprotein possessing three extracellular immunoglobulin-like domains. Among peripheral blood cells, CD96 is expressed on T cells as well as NK cells and a subpopulation of B cells. A possible function of this receptor in NK cell-mediated killing activities was suggested recently. Moreover, CD96 was described as a tumor marker for T-cell acute lymphoblastic leukemia and acute myeloid leukemia. CD96 binds to CD155 (poliovirus receptor) and nectin-1, an adhesion receptor related to CD155. Here we report that human but not mouse CD96 is expressed in two splice variants possessing either an I-like (variant 1) or V-like (variant 2) second domain. With the notable exception of an AML tumor sample, variant 2 predominates in all the CD96-expressing cell types and tissues examined. Using chimeric human/murine CD96 receptors, we show that the interaction with its ligands is mediated via the outermost V-like domain. In contrast to mouse, however, the binding of human CD96 to CD155 is sensitive to the characteristics of the two downstream domains. This is illustrated by a significantly weaker CD96/CD155 interaction mediated by variant 1 when compared with variant 2. Moreover, recent evidence suggested that mutations in human CD96 correlate with the occurrence of a rare form of trigonocephaly. One such mutation causing a single amino acid exchange in the third domain of human CD96 decreased the capacity of both variants to bind to CD155 considerably, suggesting that a CD96-driven adhesion to CD155 may be crucial in developmental processes.

The adhesion receptor CD155 determines the magnitude of humoral immune responses against orally ingested antigens.

CD155, originally known as the cellular receptor for poliovirus, is the founding member of a subfamily of immunoglobulin-like adhesion receptors. Apart from its function in establishing adherens junctions between contacting epithelial cells, the engagement of CD155 with two recently identified ligands, CD226 and CD96, mediates immunologically relevant processes such as NK cell-driven killing of tumor cells in humans. Here we report on the generation and immunological analysis of mice constitutively deficient of CD155. Moreover, the expression profile of CD155 on hematopoietic cells has been determined using newly established antibodies. CD155-deficient mice develop normally without displaying an overt phenotype. However, the animals are distinguished by distinct deficits in the development of a regular humoral immune response. Whereas systemic challenges revealed no differences, orally administered antigen evoked less efficient IgG and IgA antibody responses despite of normal IgM titers when compared to wild-type mice. Therefore, CD155 may assist in an efficient humoral immune response generated within the intestinal immune system.

Characterization and identification of Tage4 as the murine orthologue of human poliovirus receptor/CD155.

CD155 is a member of the immunoglobulin superfamily also known as the human receptor for poliovirus (PVR). Transmembrane glycoproteins related to CD155, the nectins, are well-characterized cell adhesion receptors displaying a high degree of sequence conservation across species. In contrast, CD155 belongs to the category of rapidly evolving genes wherefore a mouse CD155 gene distinguished by an affirmative extent of amino acid conservation as observed for nectins is absent. Consequently, the existing genetic evidence by itself is an inferior indicator to consider whether Tage4, a mouse orphan receptor, represents the murine orthologue of CD155. In the present study Tage4 cDNA was cloned from mouse lung and further characterized genetically. CD155 and Tage4 possess an identical genomic organization and reside in syntenic chromosomal regions. The Tage4 expression pattern was explored applying a newly generated antibody. Both receptors, CD155 in human and Tage4 in mouse, are expressed by intestinal epithelia as well as by follicle associated epithelium and follicular dendritic cells inside Peyer's patches of the gut associated lymphoid tissue. Furthermore, Tage4 lacks self-adhesion capacity but binds to vitronectin, two known features of CD155. These data indicate that Tage4 represents the functional orthologue of CD155 in mouse. Therefore, we suggest to rename Tage4 into rodent CD155.