Topological analysis of 3D digital ovules identifies cellular patterns associated with ovule shape diversity.

Tissue morphogenesis remains poorly understood. In plants, a central problem is how the 3D cellular architecture of a developing organ contributes to its final shape. We address this question through a comparative analysis of ovule morphogenesis, taking advantage of the diversity in ovule shape across angiosperms. Here, we provide a 3D digital atlas of Cardamine hirsuta ovule development at single cell resolution and compare it with an equivalent atlas of Arabidopsis thaliana. We introduce nerve-based topological analysis as a tool for unbiased detection of differences in cellular architectures and corroborate identified topological differences between two homologous tissues by comparative morphometrics and visual inspection. We find that differences in topology, cell volume variation and tissue growth patterns in the sheet-like integuments and the bulbous chalaza are associated with differences in ovule curvature. In contrast, the radialized conical ovule primordia and nucelli exhibit similar shapes, despite differences in internal cellular topology and tissue growth patterns. Our results support the notion that the structural organization of a tissue is associated with its susceptibility to shape changes during evolutionary shifts in 3D cellular architecture.

Phenotypic and functional analysis in HER2+ targeted therapy of human NK cell subpopulation according to the expression of FcεRIγ and NKG2C in breast cancer patients.

Adaptive NK cells constitute an NK cell subpopulation, which expands after human cytomegalovirus (HCMV) infection. This subpopulation has stronger production of cytokines after CD16 stimulation, longer life and persistence than conventional NK cells and are, therefore, interesting tools for cancer immunotherapy. Since there is limited information on adaptive NK cells in cancer patients, we described this population phenotypically and functionally, by flow cytometry, in the context of HER2 + breast cancer (BC) directed therapy. We assessed HCMV status in 78 patients with BC. We found that, similarly to healthy donors (HD), a high proportion of BC patients were HCMV-positive, and nearly 72% of them had an adaptive NK cell subpopulation characterized by the loss of FcεRIγ intracellular adaptor protein or the presence of NKG2C receptor. However, in BC patients, FcεRIγ- and NKG2C + NK cell populations overlapped to a lesser extent than in HD. Otherwise, no profound phenotypic differences were found between BC patients and HD. Although FcεRIγ- or NKG2C + NK cell subsets from BC patients produced more IFN-γ than their FcεRIγ + or NKG2C- NK cell counterparts, IFN-γ production increased only when NK cells simultaneously expressed FcεRIγ- and NKG2C + , whereas in HD the presence of NKG2C marker was sufficient to display greater functionality. Furthermore, in a group of patients treated with chemotherapy and Trastuzumab plus Pertuzumab, FcεRIγ-NKG2C + and FcεRIγ-NKG2C- NK cells retained greater functionality after treatment than FcεRIγ + NKG2C- NK cells. These results suggest that the presence or magnitude of adaptive NK cell subsets might serve as a key determinant for therapeutic approaches based on antibodies directed against tumor antigens.

NKG2A-checkpoint inhibition and its blockade critically depends on peptides presented by its ligand HLA-E.

NKG2A has emerged as a new immunotherapy target and its blockade with the novel immune checkpoint inhibitor (ICI) monalizumab can boost both NK cell and CD8+ T cell responses. NKG2A forms heterodimers with CD94 and binds to the human non-classical MHC class I molecule HLA-E. HLA-E forms complexes with a limited set of peptides mainly derived from the leader sequences of the classical MHC class I molecules (HLA-A, HLA-B and HLA-C) and the non-classical class I paralogue HLA-G, and it is well established that the interaction between CD94/NKG2x receptors and its ligand HLA-E is peptide-sensitive. Here, we have evaluated peptide dependence of NKG2A-mediated inhibition and the efficiency of interference by monalizumab in a transcriptional T cell reporter system. NKG2A inhibition was mediated by cell-expressed HLA-E molecules stably presenting disulfate-trapped peptide ligands. We show that different HLA-class I leader peptides mediate varying levels of inhibition. We have used NKG2A/NKG2C chimeric receptors to map the binding site of NKG2A and NKG2C blocking antibodies. Furthermore, we determined the functional EC50 values of blocking NKG2A antibodies and show that they greatly depend on the HLA-leader peptide presented by HLA-E. Monalizumab was less effective in augmenting NK cell-mediated killing of target cells displaying HLA-G peptide on HLA-E, than cells expressing HLA-E complexed with HLA-A, HLA-B and HLA-C peptides. Our results indicate that peptides displayed by HLA-E molecules on tumour cells might influence the effectivity of NKG2A-ICI therapy and potentially suggest novel approaches for patient stratification, for example, based on tumoral HLA-G levels.

HLA-E Peptide Repertoire and Dimorphism-Centerpieces in the Adaptive NK Cell Puzzle?

Adaptive Natural Killer (NK) cells, a heterogenous subpopulation of human NK cells with a unique phenotypic and functional signature, became arguably one of the central areas of interest in the field. While their existence seems closely associated with prior exposure to human cytomegalovirus (HCMV), many questions regarding their origin and regulation remain unanswered. However, a common denominator for the majority of adaptive NK cells is the expression of the activating heterodimeric receptor CD94/NKG2C that binds to HLA-E, a non-classical HLA molecule, that displays a comparably restricted expression pattern, very limited polymorphism and presents a distinct set of peptides. Recent studies suggest that-in analogy to T cell responses-peptides presented on HLA-E could play an unexpectedly decisive role for the biology of adaptive NK cells. Here, we discuss how this perspective on the CD94/NKG2C-HLA-E axis aligns with the existing literature and speculate about possible translational implication.

Distinct HLA-E Peptide Complexes Modify Antibody-Driven Effector Functions of Adaptive NK Cells.

Adaptive NK cells are characterized by profound alterations in multiple signaling molecules, transcription factors, and epigenetic modifications compared with canonical NK cells. Although their existence is associated with prior exposure to human cytomegalovirus (HCMV), key questions regarding their regulation and function remain. A large proportion of adaptive NK cells express the activating receptor CD94/NKG2C, binding to human leukocyte antigen E (HLA-E), that presents a limited set of peptides. We show that adaptive NK cells discriminate differences between HLA-E-peptide complexes with exquisite specificity. Prolonged exposure to an environment displaying the HLA-E peptide ligand VMAPRTLFL, derived from the leader sequence of HLA-G, enriched adaptive NK cells with low FcεRγ expression, upregulated CD25 expression, increased proliferative activity, and resulted in elevated antibody-dependent cellular cytotoxicity and IFN-γ responses compared with other HLA-E peptide complexes. Our study demonstrates that recognition of alterations in the HLA-E ligandome via an activating receptor can influence heterologous effector mechanisms and proliferation in adaptive NK cells.

Hepatitis C Virus and Human Cytomegalovirus-Natural Killer Cell Subsets in Persistent Viral Infections.

Hepatitis C virus (HCV) and human cytomegalovirus (HCMV) are prominent examples of RNA and DNA viruses, respectively, that establish a persistent infection in their host. HCV affects over 185 million patients worldwide, who are at high risk for developing liver fibrosis, liver cirrhosis, and ultimately hepatocellular carcinoma. Recent breakthroughs in HCV therapy, using direct-acting antivirals have provided the opportunity to monitor natural killer (NK) cells after clearance of a chronic infection. There is now increasing evidence that the individual NK cell repertoire before infection is predictive for the course of disease. HCMV affects the majority of the global population. While being asymptomatic in healthy individuals, HCMV represents a severe clinical challenge in immunocompromised patients. Both viral infections, HCV and HCMV, lead to long-lasting and profound alterations within the entire NK cell compartment. This review article, will discuss the diverse range of changes in the NK cell compartment as well as potential consequences for the course of disease.

CD2-CD58 interactions are pivotal for the activation and function of adaptive natural killer cells in human cytomegalovirus infection.

The existence and expansion of adaptive NK-cell subsets have been linked to HCMV infection. Phenotypically, a majority of adaptive NK cells expresses the activating receptor NKG2C and CD57. Some of the molecular factors driving the expansion of NKG2C+ CD57+ NK cells in HCMV infection have been identified. The direct interaction of adaptive NK cells with HCMV-infected cells, preceding the expansion, however, remains less studied. Recently, adaptive NK cells were reported to express higher levels of the co-activating receptor CD2. We explored whether CD2 was directly involved in the response of adaptive NK cells to HCMV. In a co-culture system of human PBMCs and productively infected fibroblasts, we observed an upregulation of CD69, CD25, and HLA-DR on all NK cells. However, only in adaptive NK cells was this increase largely blocked by antibodies against CD2 and CD58. Functionally, this blockade also resulted in diminished production of IFN-γ and TNF-α by adaptive human NK cells in response to HCMV-infected cells. Our results demonstrate that binding of CD2 to upregulated CD58 on infected cells is a critical event for antibody-mediated activation and subsequent effector functions of adaptive NKG2C+ CD57+ NK cells during the antiviral response.

Immune Adaptation to Environmental Influence: The Case of NK Cells and HCMV.

The immune system of an individual human is determined by heritable traits and a continuous process of adaptation to a broad variety of extrinsic, non-heritable factors such as viruses, bacteria, dietary components and more. Cytomegalovirus (CMV) successfully infects the majority of the human population and establishes latency, thereby exerting a life-long influence on the immune system of its host. CMV has been shown to influence the majority of immune parameters in healthy individuals. Here we focus on adaptive changes induced by CMV in subsets of Natural Killer (NK) cells, changes that question our very definition of adaptive and innate immunity by suggesting that adaptations of immune cells to environmental influences occur across the entire human immune system and not restricted to the classical adaptive branch of the immune system.

IL-12-producing monocytes and HLA-E control HCMV-driven NKG2C+ NK cell expansion.

Human cytomegalovirus (HCMV) infection is the most common cause of congenital viral infections and a major source of morbidity and mortality after organ transplantation. NK cells are pivotal effector cells in the innate defense against CMV. Recently, hallmarks of adaptive responses, such as memory-like features, have been recognized in NK cells. HCMV infection elicits the expansion of an NK cell subset carrying an activating receptor heterodimer, comprising CD94 and NKG2C (CD94/NKG2C), a response that resembles the clonal expansion of adaptive immune cells. Here, we determined that expansion of this NKG2C(+) subset and general NK cell recovery rely on signals derived from CD14(+) monocytes. In a coculture system, a subset of CD14(+) cells with inflammatory monocyte features produced IL-12 in response to HCMV-infected fibroblasts, and neutralization of IL-12 in this model substantially reduced CD25 upregulation and NKG2C(+) subset expansion. Finally, blockade of CD94/NKG2C on NK cells or silencing of the cognate ligand HLA-E in infected fibroblasts greatly impaired expansion of NKG2C(+) NK cells. Together, our results reveal that IL-12, CD14(+) cells, and the CD94/NKG2C/HLA-E axis are critical for the expansion of NKG2C(+) NK cells in response to HCMV infection. Moreover, strategies targeting the NKG2C(+) NK cell subset have the potential to be exploited in NK cell-based intervention strategies against viral infections and cancer.

Downregulation of the activating NKp30 ligand B7-H6 by HDAC inhibitors impairs tumor cell recognition by NK cells.

Natural killer (NK) cells are central effector cells during innate immune responses against cancer. Natural cytotoxicity receptors expressed by NK cells such as NKp30 are involved in the recognition of transformed cells. Recently, the novel B7 family member B7-H6, which is expressed on the cell surface of various tumor cells including hematological malignancies, was identified as an activating ligand for NKp30. To investigate expression and regulation of B7-H6, we generated monoclonal antibodies. Our study reveals that B7-H6 surface protein and messenger RNA (mRNA) expression in various tumor cell lines was downregulated upon treatment with pan- or class I histone deacetylase inhibitors (HDACi) as well as after small interfering RNA-mediated knockdown of the class I histone deacetylases (HDAC) 2 or 3. B7-H6 downregulation was associated with decreased B7-H6 reporter activity and reduced histone acetylation at the B7-H6 promoter. In certain primary lymphoma and hepatocellular carcinoma samples, B7-H6 mRNA levels were elevated and correlated with HDAC3 expression. Finally, downregulation of B7-H6 on tumor cells by HDACi reduced NKp30-dependent effector functions of NK cells. Thus, we identified a novel mechanism that governs B7-H6 expression in tumor cells that has implications for potential cancer treatments combining immunotherapy with HDACi.

Dendritic cells in cytomegalovirus infection: viral evasion and host countermeasures.

Human cytomegalovirus (HCMV) is a beta-herpesvirus that infects the majority of the population during early childhood and thereafter establishes life-long latency. Primary infection as well as spontaneous reactivation usually remains asymptomatic in healthy hosts but can, in the context of systemic immunosuppression, result in substantial morbidity and mortality. HCMV counteracts the host immune response by interfering with the recognition of infected cells. A growing body of literature has also suggested that the virus evades the immune system by paralyzing the initiators of antiviral immune responses--the dendritic cells (DCs). In the current review, we discuss the effects of CMV (HCMV and murine CMV) on various DC subsets and the ensuing innate and adaptive immune responses. The impact of HCMV on DCs has mainly been investigated using monocyte-derived DCs, which are rendered functionally impaired by infection. In mouse models, DCs are targets of viral evasion as well, but the complex cross-talk between DCs and natural killer cells has, however, demonstrated an instrumental role for DCs in the control and clearance of viral infection. Fewer studies address the role of peripheral blood DC subsets, plasmacytoid DCs and CD11c+ myeloid DCs in the response against HCMV. These DCs, rather than being paralyzed by HCMV, are largely resistant to infection, mount a vigorous first-line defense and induce T-cell responses to the virus. This possibly provides a partial explanation for an intriguing conundrum: the highly efficient control of viral infection and reactivation in immunocompetent hosts in spite of multi-layered viral evasion mechanisms.

Structural elements underlying the high binding affinity of human cytomegalovirus UL18 to leukocyte immunoglobulin-like receptor-1.

Human cytomegalovirus (HCMV) encodes UL18, a major histocompatibility complex (MHC) class I homologue that binds to the leukocyte immunoglobulin-like receptor (LIR)-1 (also called ILT2/CD85j/LILRB1), an inhibitory receptor expressed on myeloid and lymphoid immune cells. The molecular basis underlying the high affinity binding of UL18 to LIR-1, compared to MHC class I molecules (MHC-I), is unclear. Based on a comparative structural analysis of a molecular model of UL18 with the crystal structure of the HLA-A2/LIR-1 complex, we identified three regions in UL18 influencing interaction with LIR-1. Comparison of the relative binding affinities of mutated UL18 proteins to LIR-1 demonstrated the importance of specific residues in each region. Substitution of residues K42/A43 and Q202, localized in the alpha1 and alpha3 domains, respectively, reduced binding affinity to LIR-1 nearly by half. The model also suggested the formation of an additional disulfide bridge in the alpha3 domain of UL18 between residues C240 and C255, not present in MHC-I. Substitution of either cysteine residue prevented association of UL18 to beta2m, abolishing binding to LIR-1. All observed differences in binding affinities translated directly into functional consequences in terms of inhibition of IFN-gamma production by T cells, mediated through the UL18-LIR-1 interaction. The larger amount of interacting regions, combined with an increased stability of the alpha3 and beta2m domains allow a higher recognition affinity of UL18 by LIR-1.

Effects of human cytomegalovirus infection on ligands for the activating NKG2D receptor of NK cells: up-regulation of UL16-binding protein (ULBP)1 and ULBP2 is counteracted by the viral UL16 protein.

Human CMV (HCMV) interferes with NK cell functions at various levels. The HCMV glycoprotein UL16 binds some of the ligands recognized by the NK-activating receptor NKG2D, namely UL16-binding proteins (ULBP) 1 and 2 and MHC class I-related chain B, possibly representing another mechanism of viral immune escape. This study addressed the expression and function of these proteins in infected cells. HCMV induced the expression of all three ULBPs, which were predominantly localized in the endoplasmic reticulum of infected fibroblasts together with UL16. However, while at a lower viral dose ULBP1 and 2 surface expression was completely inhibited compared to ULBP3, at a higher viral dose cell surface expression of ULBP1 and ULBP2 was delayed. The induction of ULBPs correlated with an increased dependency on NKG2D for recognition; however, the overall NK sensitivity did not change (suggesting that additional viral mechanisms interfere with NKG2D-independent pathways for recognition). Infection with a UL16 deletion mutant virus resulted in a different pattern compared to the wild type: all three ULBP molecules were induced with similar kinetics at the cell surface, accompanied by a pronounced, entirely NKG2D-dependent increase in NK sensitivity. Together our findings show that upon infection with HCMV, the host cell responds by expression of ULBPs and increased susceptibility to the NKG2D-mediated component of NK cell recognition, but UL16 limits these effects by interfering with the surface expression of ULBP1 and ULBP2.