Mast cells expedite control of pulmonary murine cytomegalovirus infection by enhancing the recruitment of protective CD8 T cells to the lungs.

The lungs are a noted predilection site of acute, latent, and reactivated cytomegalovirus (CMV) infections. Interstitial pneumonia is the most dreaded manifestation of CMV disease in the immunocompromised host, whereas in the immunocompetent host lung-infiltrating CD8 T cells confine the infection in nodular inflammatory foci and prevent viral pathology. By using murine CMV infection as a model, we provide evidence for a critical role of mast cells (MC) in the recruitment of protective CD8 T cells to the lungs. Systemic infection triggered degranulation selectively in infected MC. The viral activation of MC was associated with a wave of CC chemokine ligand 5 (CCL5) in the serum of C57BL/6 mice that was MC-derived as verified by infection of MC-deficient Kit(W-sh/W-sh) "sash" mutants. In these mutants, CD8 T cells were recruited less efficiently to the lungs, correlating with enhanced viral replication and delayed virus clearance. A causative role for MC was verified by MC reconstitution of "sash" mice restoring both, efficient CD8 T-cell recruitment and infection control. These results reveal a novel crosstalk axis between innate and adaptive immune defense against CMV, and identify MC as a hitherto unconsidered player in the immune surveillance at a relevant site of CMV disease.

An endocytic YXXΦ (YRRF) cargo sorting motif in the cytoplasmic tail of murine cytomegalovirus AP2 'adapter adapter' protein m04/gp34 antagonizes virus evasion of natural killer cells.

Viruses have evolved proteins that bind immunologically relevant cargo molecules at the cell surface for their downmodulation by internalization. Via a tyrosine-based sorting motif YXXΦ in their cytoplasmic tails, they link the bound cargo to the cellular adapter protein-2 (AP2), thereby sorting it into clathrin-triskelion-coated pits for accelerated endocytosis. Downmodulation of CD4 molecules by lentiviral protein NEF represents the most prominent example. Based on connecting cargo to cellular adapter molecules, such specialized viral proteins have been referred to as 'connectors' or 'adapter adapters.' Murine cytomegalovirus glycoprotein m04/gp34 binds stably to MHC class-I (MHC-I) molecules and suspiciously carries a canonical YXXΦ endocytosis motif YRRF in its cytoplasmic tail. Disconnection from AP2 by motif mutation ARRF should retain m04-MHC-I complexes at the cell surface and result in an enhanced silencing of natural killer (NK) cells, which recognize them via inhibitory receptors. We have tested this prediction with a recombinant virus in which the AP2 motif is selectively destroyed by point mutation Y248A, and compared this with the deletion of the complete protein in a Δm04 mutant. Phenotypes were antithetical in that loss of AP2-binding enhanced NK cell silencing, whereas absence of m04-MHC-I released them from silencing. We thus conclude that AP2-binding antagonizes NK cell silencing by enhancing endocytosis of the inhibitory ligand m04-MHC-I. Based on a screen for tyrosine-based endocytic motifs in cytoplasmic tail sequences, we propose here the new hypothesis that most proteins of the m02-m16 gene family serve as 'adapter adapters,' each selecting its specific cell surface cargo for clathrin-assisted internalization.

Mast cells: innate attractors recruiting protective CD8 T cells to sites of cytomegalovirus infection.

Reactivation of latent cytomegalovirus (CMV) in the transient immunocompromised state after hematoablative treatment is a major concern in patients undergoing hematopoietic cell transplantation (HCT) as a therapy of hematopoietic malignancies. Timely reconstitution of antiviral CD8 T cells and their efficient recruitment to the lungs is crucial for preventing interstitial pneumonia, the most severe disease manifestation of CMV in HCT recipients. Here, we review recent work in a murine model, implicating mast cells (MC) in the control of pulmonary infection. Murine CMV (mCMV) productively infects MC in vivo and triggers their degranulation, resulting in the release of the CC chemokine ligand 5 (CCL5) that attracts CD8 T cells to infiltrate infected tissues. Comparing infection of MC-sufficient C57BL/6 mice and congenic MC-deficient Kit (W-sh/W-sh) "sash" mutants revealed an inverse relation between the number of lung-infiltrating CD8 T cells and viral burden in the lungs. Specifically, reduced lung infiltration by CD8 T cells in "sash" mutants was associated with an impaired infection control. The causal, though indirect, involvement of MC in antiviral control was confirmed by reversion of the deficiency phenotype in "sash" mutants reconstituted with MC. These recent findings predict that efficient MC reconstitution facilitates the control of CMV infection also in immunocompromised HCT recipients.

The NK cell response to mouse cytomegalovirus infection affects the level and kinetics of the early CD8(+) T-cell response.

Natural killer (NK) cells and CD8(+) T cells play a prominent role in the clearance of mouse cytomegalovirus (MCMV) infection. The role of NK cells in modulating the CD8(+) T-cell response to MCMV infection is still the subject of intensive research. For analyzing the impact of NK cells on mounting of a CD8(+) T-cell response and the contribution of these cells to virus control during the first days postinfection (p.i.), we used C57BL/6 mice in which NK cells are specifically activated through the Ly49H receptor engaged by the MCMV-encoded ligand m157. Our results indicate that the requirement for CD8(+) T cells in early MCMV control inversely correlates with the engagement of Ly49H. While depletion of CD8(+) T cells has only a minor effect on the early control of wild-type MCMV, CD8(+) T cells are essential in the control of Δm157 virus. The frequencies of virus epitope-specific CD8(+) T cells and their activation status were higher in mice infected with Δm157 virus. In addition, these mice showed elevated levels of alpha interferon (IFN-α) and several other proinflammatory cytokines as early as 1.5 days p.i. Although the numbers of conventional dendritic cells (cDCs) were reduced later during infection, particularly in Δm157-infected mice, they were not significantly affected at the peak of the cytokine response. Altogether, we concluded that increased antigen load, preservation of early cDCs' function, and higher levels of innate cytokines collectively account for an enhanced CD8(+) T-cell response in C57BL/6 mice infected with a virus unable to activate NK cells via the Ly49H-m157 interaction.

The roles of different forms of IL-15 in human melanoma progression.

Background: Melanoma is a lethal skin cancer, and the risk of developing it is increased by exposure to ultraviolet (UV) radiation. The production of cytokines such as interleukin-15 (IL-15), induced by the exposure of skin cells to UV rays, could also promote melanoma development. The aim of this study is to investigate the possible role of Interleukin-15/Interleukin-15 Receptor α (IL-15/IL-15Rα) complexes in melanoma development. Methods: The expression of IL-15/IL-15Rα complexes by melanoma cells was evaluated both ex vivo and in vitro by tissue microarray, PCR, and flow cytometry. The presence of the soluble complex (sIL-15/IL-15Rα) in the plasma of metastatic melanoma patients was detected using an ELISA assay. Subsequently, we investigated the impact of natural killer (NK) cell activation after rIL-2 starvation followed by exposure to the sIL-15/IL-15Rα complex. Finally, by analyzing public datasets, we studied the correlation between IL-15 and IL-15Rα expressions and melanoma stage, NK and T-cell markers, and overall survival (OS). Results: Analysis of a melanoma tissue microarray shows a significant increase in the number of IL-15+ tumor cells from the benign nevi to metastatic melanoma stages. Metastatic melanoma cell lines express a phorbol-12-myristate-13-acetate (PMA)-cleavable membrane-bound IL-15 (mbIL-15), whereas cultures from primary melanomas express a PMA-resistant isoform. Further analysis revealed that 26% of metastatic patients present with consistently high plasmatic levels of sIL-15/IL-15Rα. When the recombinant soluble human IL-15/IL-15Rα complex is added to briefly starved rIL-2-expanded NK cells, these cells exhibit strongly reduced proliferation and levels of cytotoxic activity against K-562 and NALM-18 target cells. The analysis of public gene expression datasets revealed that high IL-15 and IL-15Rα intra-tumoral production correlates with the high levels of expression of CD5+ and NKp46+ (T and NK markers) and significantly correlates with a better OS in stages II and III, but not in stage IV. Conclusions: Membrane-bound and secreted IL-15/IL-15Rα complexes are continuously present during progression in melanoma. It is notable that, although IL-15/IL-15Rα initially promoted the production of cytotoxic T and NK cells, at stage IV promotion of the development of anergic and dysfunctional cytotoxic NK cells was observed. In a subgroup of melanoma metastatic patients, the continuous secretion of high amounts of the soluble complex could represent a novel NK cell immune escape mechanism.

Linoleic acid potentiates CD8+ T cell metabolic fitness and antitumor immunity.

The metabolic state represents a major hurdle for an effective adoptive T cell therapy (ACT). Indeed, specific lipids can harm CD8+ T cell (CTL) mitochondrial integrity, leading to defective antitumor responses. However, the extent to which lipids can affect the CTL functions and fate remains unexplored. Here, we show that linoleic acid (LA) is a major positive regulator of CTL activity by improving metabolic fitness, preventing exhaustion, and stimulating a memory-like phenotype with superior effector functions. We report that LA treatment enhances the formation of ER-mitochondria contacts (MERC), which in turn promotes calcium (Ca2+) signaling, mitochondrial energetics, and CTL effector functions. As a direct consequence, the antitumor potency of LA-instructed CD8 T cells is superior in vitro and in vivo. We thus propose LA treatment as an ACT potentiator in tumor therapy.

Immune control in the absence of immunodominant epitopes: implications for immunotherapy of cytomegalovirus infection with antiviral CD8 T cells.

Adoptive transfer of virus-specific donor-derived CD8 T cells is a therapeutic option to prevent cytomegalovirus (CMV) disease in recipients of hematopoietic cell transplantation. Due to their high coding capacity, human as well as animal CMVs have the potential to encode numerous CD8 T cell epitopes. Although the CD8 T cell response to CMVs is indeed broadly specific in that it involves epitopes derived from almost every open reading frame when tested for cohorts of immune CMV carriers representing the polymorphic MHC/HLA distribution in the population, the response in any one individual is directed against relatively few epitopes selected by the private combination of MHC/HLA alleles. Of this individually selected set of epitopes, few epitopes are 'immunodominant' in terms of magnitude of the response directed against them, while others are 'subdominant' according to this definition. In the assumption that 'immunodominance' indicates 'relevance' in antiviral control, research interest was focused on the immunodominant epitopes (IDEs) and their potential use in immunotherapy and in vaccines. The murine model has provided 'proof of concept' for the efficacy of CD8 T cell therapy of CMV infection. By experimental modulation of the CD8 T cell 'immunome' of murine CMV constructing an IDE deletion mutant, we have used this established cytoimmunotherapy model (a) for evaluating the actual contribution of IDEs to the control of infection and (b) for answering the question whether antigenicity-determining codon polymorphisms in IDE-encoding genes of CMV strains impact on the efficacy of CD8 T cell immunotherapy in case the donor and the recipient harbor different CMV strains.

Murine cytomegalovirus immune evasion proteins operative in the MHC class I pathway of antigen processing and presentation: state of knowledge, revisions, and questions.

Medical interest in cytomegalovirus (CMV) is based on lifelong neurological sequelae, such as sensorineural hearing loss and mental retardation, resulting from congenital infection of the fetus in utero, as well as on CMV disease with multiple organ manifestations and graft loss in recipients of hematopoietic cell transplantation or solid organ transplantation. CMV infection of transplantation recipients occurs consequent to reactivation of virus harbored in a latent state in the transplanted donor cells and tissues, or in the tissues of the transplantation recipient herself or himself. Hence, CMV infection is a paradigm for a viral infection that causes disease primarily in the immunocompromised host, while infection of the immunocompetent host is associated with only mild and nonspecific symptoms so that it usually goes unnoticed. Thus, CMV is kept under strict immune surveillance. These medical facts are in apparent conflict with the notion that CMVs in general, human CMV as well as animal CMVs, are masters of 'immune evasion', which during virus-host co-speciation have convergently evolved sophisticated mechanisms to avoid their recognition by innate and adaptive immunity of their respective host species, with viral genes apparently dedicated to serve just this purpose (Reddehase in Nat Rev Immunol 2:831-844, 2002). With focus on viral interference with antigen presentation to CD8 T cells in the preclinical model of murine CMV infection, we try here to shed some more light on the in vivo balance between host immune surveillance of CMV infection and viral 'immune evasion' strategies.

Parameters determining the efficacy of adoptive CD8 T-cell therapy of cytomegalovirus infection.

Reactivation of latent cytomegalovirus (CMV) in the transient state of immunodeficiency after hematopoietic cell transplantation (HCT) is the most frequent and severe viral complication endangering leukemia therapy success. By infecting the bone marrow (BM) stroma of the transplantation recipient, CMV can directly interfere with BM repopulation by the transplanted donor-derived hematopoietic cells and thus delay immune reconstitution of the recipient. Cytopathogenic virus spread in tissues can result in CMV disease with multiple organ manifestations of which interstitial pneumonia is the most feared. There exists a 'window of risk' between hematoablative treatment and reconstitution of antiviral immunity after HCT, whereby timely reconstitution of antiviral CD8 T cells is a recognized positive prognostic parameter for the control of reactivated CMV infection and prevention of CMV disease. Supplementation of endogenous reconstitution by adoptive cell transfer of 'ready-to-go' effector and/or memory virus epitope-specific CD8 T cells is a therapeutic option to bridge the 'window of risk.' Preclinical research in murine models of CMV disease has been pivotal by providing 'proof of concept' for a benefit from CD8 T-cell therapy of HCT-associated CMV disease (reviewed in Holtappels et al. Med Microbiol Immunol 197:125-134, 2008). Here, we give an update of our previous review with focus on parameters that determine the efficacy of adoptive immunotherapy of CMV infection by antiviral CD8 T cells in the murine model.

Antigen-presenting cells of haematopoietic origin prime cytomegalovirus-specific CD8 T-cells but are not sufficient for driving memory inflation during viral latency.

Expansion of the CD8 T-cell memory pool, also known as 'memory inflation', for certain but not all viral epitopes in latently infected host tissues is a special feature of the immune response to cytomegalovirus. The L(d)-presented murine cytomegalovirus (mCMV) immediate-early (IE) 1 peptide is the prototype of an epitope that is associated with memory inflation. Based on the detection of IE1 transcripts in latently infected lungs it was previously proposed that episodes of viral gene expression and antigenic activity due to desilencing of a limited number of viral genes may drive epitope-specific memory inflation. This would imply direct antigen presentation through latently infected host tissue cells rather than cell death-associated cross-presentation of viral antigens derived from productively infected cells through uninfected, professional antigen-presenting cells (profAPCs). To address the role of bone marrow-derived profAPCs in CD8 T-cell priming and memory to mCMV, we have used here a combined sex-mismatched and MHC class-I mismatched dual-marker bone marrow chimera model in which presentation of the IE1 epitope is restricted to donor-derived sry(+)L(d+) cells of haematopoietic differentiation lineages. Successful CD8 T-cell priming specific for the L(d)- and D(d)-presented inflationary epitopes IE1 and m164, respectively, but selective failure in IE1 epitope-specific memory inflation in these chimeras indicates different modes of antigen presentation involved in CD8 T-cell priming and memory inflation. These data suggest that memory inflation during mCMV latency requires expression of the epitope-presenting MHC class-I molecule by latently infected non-haematopoietic host tissue cells and thus predicts a role for direct antigen presentation in memory inflation.

Pediatric Tumors-Mediated Inhibitory Effect on NK Cells: The Case of Neuroblastoma and Wilms' Tumors.

Natural killer (NK) cells play a key role in the control of cancer development, progression and metastatic dissemination. However, tumor cells develop an array of strategies capable of impairing the activation and function of the immune system, including NK cells. In this context, a major event is represented by the establishment of an immunosuppressive tumor microenvironment (TME) composed of stromal cells, myeloid-derived suppressor cells, tumor-associated macrophages, regulatory T cells and cancer cells themselves. The different immunoregulatory cells infiltrating the TME, through the release of several immunosuppressive molecules or by cell-to-cell interactions, cause an impairment of the recruitment of NK cells and other lymphocytes with effector functions. The different mechanisms by which stromal and tumor cells impair NK cell function have been particularly explored in adult solid tumors and, in less depth, investigated and discussed in a pediatric setting. In this review, we will compare pediatric and adult solid malignancies concerning the respective mechanisms of NK cell inhibition, highlighting novel key data in neuroblastoma and Wilms' tumor, two of the most frequent pediatric extracranial solid tumors. Indeed, both tumors are characterized by the presence of stromal cells acting through the release of immunosuppressive molecules. In addition, specific tumor cell subsets inhibit NK cell cytotoxic function by cell-to-cell contact mechanisms likely controlled by the transcriptional coactivator TAZ. These findings could lead to a more performant diagnostic approach and to the development of novel immunotherapeutic strategies targeting the identified cellular and molecular targets.

A novel transmembrane domain mediating retention of a highly motile herpesvirus glycoprotein in the endoplasmic reticulum.

Gene m164 of murine cytomegalovirus belongs to the large group of 'private' genes that show no homology to those of other cytomegalovirus species and are thought to represent 'host adaptation' genes involved in virus-host interaction. Previous interest in the m164 gene product was based on the presence of an immunodominant CD8 T-cell epitope presented at the surface of infected cells, despite interference by viral immune-evasion proteins. Here, we provide data to reveal that the m164 gene product shows unusual features in its cell biology. A novel strategy of mass-spectrometric analysis was employed to map the N terminus of the mature protein, 107 aa downstream of the start site of the predicted open reading frame. The resulting 36.5 kDa m164 gene product is identified here as an integral type-I membrane glycoprotein with exceptional intracellular trafficking dynamics, moving within the endoplasmic reticulum (ER) and outer nuclear membrane with an outstandingly high lateral membrane motility, actually 100 times higher than those published for cellular ER-resident proteins. Notably, gp36.5/m164 does not contain any typical ER-retention/retrieval signals, such as the C-terminal motifs KKXX or KXKXX, and does not pass the Golgi apparatus. Instead, it belongs to the rare group of viral glycoproteins in which the transmembrane domain (TMD) itself mediates direct ER retention. This is the first report relating TMD usage of an ER-resident transmembrane protein to its lateral membrane motility as a paradigm in cell biology. We propose that TMD usage for ER retention facilitates free and fast floating in ER-related membranes and between ER subdomains.

Cognate Nonlytic Interactions between CD8+ T Cells and Breast Cancer Cells Induce Cancer Stem Cell-like Properties.

Targeting of tumor immune escape mechanisms holds enormous therapeutic potential. Still, most patients progress under immune checkpoint blockade and some even become hyperprogressors. To investigate how cancer cells respond to activated but ineffective T cells, we challenged peptide-loaded MCF-7 breast cancer cells with antigen-specific CD8+ T cells in which lytic granules had been destroyed by pretreatment with Concanamycin A. Gene expression analysis after coculture revealed simultaneous induction of PD-L1, IDO1, CEACAM1, and further immunoregulatory checkpoints in breast cancer cells. Strikingly, we further observed gene signatures characteristic for dedifferentiation and acquisition of pluripotency markers including Yamanaka factors. Cognate interaction with nonlytic CD8+ T cells also increased the proportion of stem cell-like cancer cells in a cell-to-cell contact- or (at least) proximity-dependent manner in various cell lines and in primary breast cancer cell cultures; this induction of stem cell-like properties was confirmed by enhanced tumor-forming capacity in immunodeficient mice. Resulting tumors were characterized by enhanced cell density, higher proliferation rates, and increased propensity for lymphoid metastasis. These findings describe a widely underappreciated pathway for immune escape, namely immune-mediated dedifferentiation of breast cancer cells, which is associated with profound changes in gene expression and cellular behavior. As the enhanced malignant potential of cancer cells after nonlytic cognate interactions with CD8+ T cells enables increased tumor growth and metastasis in BALB/cnu/nu mice, the described mechanism may provide a possible explanation for the clinical phenomenon of hyperprogression in response to unsuccessful immunotherapy. SIGNIFICANCE: This study shows that ineffective immune responses not only fail to clear a malignancy, but can also activate pathways in cancer cells that promote stemness and tumor-seeding capacity.

Reconstitution of CD8 T Cells Protective against Cytomegalovirus in a Mouse Model of Hematopoietic Cell Transplantation: Dynamics and Inessentiality of Epitope Immunodominance.

Successful reconstitution of cytomegalovirus (CMV)-specific CD8(+) T cells by hematopoietic cell transplantation (HCT) gives a favorable prognosis for the control of CMV reactivation and prevention of CMV disease after hematoablative therapy of hematopoietic malignancies. In the transient immunocompromised state after HCT, pre-emptive cytoimmunotherapy with viral epitope-specific effector or memory CD8(+) T cells is a promising option to speed up antiviral control. Despite high-coding capacity of CMVs and a broad CD8(+) T-cell response on the population level, which reflects polymorphism in major histocompatibility complex class-I (MHC-I) glycoproteins, the response in terms of quantity of CD8(+) T cells in any individual is directed against a limited set of CMV-encoded epitopes selected for presentation by the private repertoire of MHC-I molecules. Such epitopes are known as "immunodominant" epitopes (IDEs). Besides host immunogenetics, genetic variance in CMV strains harbored as latent viruses by an individual HCT recipient can also determine the set of IDEs, which complicates a "personalized immunotherapy." It is, therefore, an important question if IDE-specific CD8(+) T-cell reconstitution after HCT is critical or dispensable for antiviral control. As viruses with targeted mutations of IDEs cannot be experimentally tested in HCT patients, we employed the well-established mouse model of HCT. Notably, control of murine CMV (mCMV) after HCT was comparably efficient for IDE-deletion mutant mCMV-Δ4IDE and the corresponding IDE-expressing revertant virus mCMV-Δ4IDE-rev. Thus, antigenicity-loss mutations in IDEs do not result in loss-of-function of a polyclonal CD8(+) T-cell population. Although IDE deletion was not associated with global changes in the response to non-IDE epitopes, the collective of non-IDE-specific CD8(+) T-cells infiltrates infected tissue and confines infection within nodular inflammatory foci. We conclude from the model, and predict also for human CMV, that there is no need to exclusively aim for IDE-specific immunoreconstitution.

Mast cells as rapid innate sensors of cytomegalovirus by TLR3/TRIF signaling-dependent and -independent mechanisms.

The succinct metaphor, 'the immune system's loaded gun', has been used to describe the role of mast cells (MCs) due to their storage of a wide range of potent pro-inflammatory and antimicrobial mediators in secretory granules that can be released almost instantly on demand to fight invaders. Located at host-environment boundaries and equipped with an arsenal of pattern recognition receptors, MCs are destined to be rapid innate sensors of pathogens penetrating endothelial and epithelial surfaces. Although the importance of MCs in antimicrobial and antiparasitic defense has long been appreciated, their role in raising the alarm against viral infections has been noted only recently. Work on cytomegalovirus (CMV) infection in the murine model has revealed MCs as players in a novel cross-talk axis between innate and adaptive immune surveillance of CMV, in that infection of MCs, which is associated with MC degranulation and release of the chemokine CCL5, enhances the recruitment of protective CD8 T cells to extravascular sites of virus replication, specifically to lung interstitium and alveolar epithelium. Here, we have expanded on these studies by investigating the conditions for MC activation and the consequent degranulation in response to host infection. Surprisingly, the data revealed two temporally and mechanistically distinct waves of MC activation: an almost instant indirect activation that depended on TLR3/TRIF signaling and delayed activation by direct infection of MCs that did not involve TLR3/TRIF signaling. Cell type-specific Cre-recombination that yielded eGFP-expressing reporter virus selectively originating from MCs identified MC as a new in vivo, first-hit target cell of productive murine CMV infection.

Reconstitution of cyanogenesis in barley (Hordeum vulgare L.) and its implications for resistance against the barley powdery mildew fungus.

Barley (Hordeum vulgare L.) produces a leucine-derived cyanogenic beta-D-glucoside, epiheterodendrin that accumulates specifically in leaf epidermis. Barley leaves are not cyanogenic, i.e. they do not possess the ability to release hydrogen cyanide, because they lack a cyanide releasing beta-D-glucosidase. Cyanogenesis was reconstituted in barley leaf epidermal cells through single cell expression of a cDNA encoding dhurrinase-2, a cyanogenic beta-D-glucosidase from sorghum. This resulted in a 35-60% reduction in colonization rate by an obligate parasite Blumeria graminis f. sp. hordei, the causal agent of barley powdery mildew. A database search for barley homologues of dhurrinase-2 identified a (1,4)-beta-D-glucan exohydrolase isozyme betaII that is located in the starchy endosperm of barley grain. The purified barley (1,4)-beta-D-glucan exohydrolase isozyme betaII was found to hydrolyze the cyanogenic beta-D-glucosides, epiheterodendrin and dhurrin. Molecular modelling of its active site based on the crystal structure of linamarase from white clover, demonstrated that the disposition of the catalytic active amino acid residues was structurally conserved. Epiheterodendrin stimulated appressoria and appressorial hook formation of B. graminis in vitro, suggesting that loss of cyanogenesis in barley leaves has enabled the fungus to utilize the presence of epiheterodendrin to facilitate host recognition and to establish infection.