Inga Ravens

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.

Human [gamma][delta] T cells are quickly reconstituted after stem-cell transplantation and show adaptive clonal expansion in response to viral infection

To investigate how the human γδ T cell pool is shaped during ontogeny and how it is regenerated after transplantation of hematopoietic stem cells (HSCs), we applied an RNA-based next-generation sequencing approach to monitor the dynamics of the repertoires of γδ T cell antigen receptors (TCRs) before and after transplantation in a prospective cohort study. We found that repertoires of rearranged genes encoding γδ TCRs (TRG and TRD) in the peripheral blood of healthy adults were stable over time. Although a large fraction of human TRG repertoires consisted of public sequences, the TRD repertoires were private. In patients undergoing HSC transplantation, γδ T cells were quickly reconstituted; however, they had profoundly altered TCR repertoires. Notably, the clonal proliferation of individual virus-reactive γδ TCR sequences in patients with reactivation of cytomegalovirus revealed strong evidence for adaptive anti-viral γδ T cell immune responses.

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.

Distinct gene expression patterns correlate with developmental and functional traits of iNKT subsets

Invariant natural killer T (iNKT) cells comprise a subpopulation of innate lymphocytes developing in thymus. A new model proposes subdividing murine iNKT cells into iNKT1, 2 and 17 cells. Here, we use transcriptome analyses of iNKT1, 2 and 17 subsets isolated from BALB/c and C57BL/6 thymi to identify candidate genes that may affect iNKT cell development, migration or function. We show that Fcɛr1γ is involved in generation of iNKT1 cells and that SerpinB1 modulates frequency of iNKT17 cells. Moreover, a considerable proportion of iNKT17 cells express IL-4 and IL-17 simultaneously. The results presented not only validate the usefulness of the iNKT1/2/17-concept but also provide new insights into iNKT cell biology.

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.

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.

CD155/CD226-interaction impacts on the generation of innate CD8(+) thymocytes by regulating iNKT-cell differentiation.

The cell surface receptor CD155 influences a variety of immune processes by binding to its ligands CD226, CD96, or TIGIT. Here, we report that the interaction of CD155 with CD226 in the thymus of BALB/c mice has a dual function. It directly influences the dwell time of memory‐like CD8+ T cells, while it is indirectly involved in generating these cells. It was shown earlier that a massive emergence of memory‐like CD8 T cells in thymus crucially depends on abundant IL‐4, secreted in steady state by iNKT2 (where iNKT is invariant NKT) cells, a subclass of iNKT cells. Here, we show that absence of either CD155 or CD226 in BALB/c mice causes a profound shift in the iNKT subtype composition in thymus, expanding the frequency and numbers of iNKT1 cells at the expense of iNKT2 cells, as well as iNKT17 cells. This shift results in a drop of available IL‐4 and creates a scenario similar to that observed in C57BL/6 mice, where iNKT1 cells predominate and iNKT2 cells are much less frequent when compared with BALB/c mice. Yet also in C57BL/6 mice, lack of CD155 or CD226 provokes a further decline in iNKT2 cells, suggesting that the observed effects are not restricted to a particular inbred strain.