Sebastian Seth

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.

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.

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.

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 (TFH) 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 TFH cells in their Peyers patches. This was paralleled by a decreased frequency of TFH cells in the GC. Moreover, the CD155 ligand CD226, which is involved in T‐cell activation, is down‐regulated during TFH cell differentiation, resulting in a complete absence of CD226 on those TFH cells residing in the GC. Concurrently, the expression of TIGIT/WUCAM, a newly discovered CD155 ligand, is induced in TFH cells. Thus, these cells replace an activating by a putative inhibitory CD155‐binding partner during their differentiation.

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.

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.

Antigen‐dependent rescue of nose‐associated lymphoid tissue (NALT) development independent of LTβR 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 Peyers 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 β receptor (LTβR). 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‐βR 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 LTβR–CXCR5 signaling axis.

Absence of CD155 aggravates acute graft-versus-host disease

In a recent paper, Nabekura et al. (1) provided compelling evidence that abrogating DNAM-1 (DNAX accessory molecule-1; CD226) activity resulted in milder development of graft-versus-host disease (GVHD). This is expected, because the role of CD226 as an important costimulatory receptor during T-cell activation is well-documented (2, 3). As shown in the paper (1), CD226 present on donor T cells contributed to the typical syndromes of GVHD. A milder course of GVHD was also achieved by treating transplanted mice with an antibody neutralizing CD226. The authors assumed that CD226 signaling is initiated by interaction of the allogeneic T cells with host cells expressing either CD112 or CD155, the two known ligands of CD226 (1). We analyzed the development of GVHD in mice deficient for CD155. Unexpectedly, the mice succumbed to GVHD within 1 wk, whereas WT mice survived for approximately 3 wk (Fig. 1A). Additional experiments suggested that the observed aggravation in the course of disease is mainly caused by CD4+ donor T cells (Fig. 1A). Additional analyses aimed at identifying the cause of premature death of CD155−/− recipients failed to reveal any differences to WT controls (Fig. 1B). Serum cytokine levels of IFNγ, IL-6, and TNFα as well as the extent of T-cell proliferation were virtually identical when investigated 3 d posttransplantation. We also subjected the intestine of recipients to a detailed histological examination 6 d posttransplantation, but again, significant differences in the degree of injury or infiltrating T cells between WT and CD155−/− mice were not detected. Of note, CD155−/− mice receiving T cell-depleted bone marrow only developed lethal GVHD after T cells differentiated from donor marrow. This corroborated that CD155−/− mice died of deleterious T-cell effects and not because of constitutional defects imposed by CD155 deficiency (Fig. 1A). Fig. 1 (A) After lethal irradiation, WT BALB/c or CD155−/− (Pvrtm1Gbn) BALB/c (KO) mice received 5 × 106 C57BL/6 T cell-depleted bone marrow cells (BM) alone (n = 8) or BM supplemented with 1 × 106 T cells (CD4+ and CD8+ T cells ... Even if the exact cause of the exacerbated course of GVHD in CD155−/− recipients remains elusive, our results suggest that presence of CD155 may attenuate otherwise devastating consequences of GVHD. Because the study by Nabekura et al. (1) clearly showed that CD226-triggered T-cell coactivation contributes to a worsening course of GVHD, CD155 possibly plays a Janus-faced role in GVHD development: its presence exerts protective effects but at the same time, aggravates GVHD by activating donor cells expressing CD226. Alternatively, as already discussed by Nabekura et al. (1), CD112 expressed by host cells in liver and intestine may be largely responsible for the observed CD226 effects. Future studies involving CD112−/− mice will help to clarify this point. Moreover, the GVHD model in use also matters. Whereas Nabekura et al. (1) chose an experimental strategy eliciting GVHD preferentially by CD8+ T cells, our investigations were based on an MHC fully mismatched model where CD4+ T cells mainly contribute to GVHD development (4). Nevertheless, we were unable to prolong survival time of CD155−/− recipients by treating mice with a CD226 neutralizing antibody (Fig. 1A) (5). Despite its importance in CD4+ T-cell stimulation and differentiation (3), CD226 may, thus, not always play a major role in the complex pathophysiology of GVHD.

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.