Magnitude of Type I Interferon Responses by Plasmacytoid Dendritic Cells After TLR7 Stimulation Is Associated With Human Immunodeficiency Virus Type 1 (HIV-1) Reservoir Sizes in Cisgender Women With HIV-1 on Antiretroviral Therapy.

Human immunodeficiency virus type 1 (HIV-1) disease manifestations differ between cisgender women and men, including better control of viral replication during primary infection and less frequent residual HIV-1 replication on antiretroviral therapy (ART) in cisgender women with HIV-1 (WWH). Investigating plasmacytoid dendritic cell (pDC) functions and HIV-1 reservoir sizes in 20 WWH on stable ART, we observed inverse correlations between interferon-α and tumor necrosis factor responses of pDCs to Toll-like receptor 7/8 stimulation and intact/total proviral HIV-1 DNA levels. Additionally, ISG15 mRNA levels in peripheral blood mononuclear cells correlated with cytokine responses of pDCs. These findings demonstrate an association between higher type I interferon responses and lower HIV-1 reservoir sizes in WWH on ART, warranting studies to identify the underlying mechanisms.

A liquid chromatography-tandem mass spectrometry based method for the quantification of adenosine nucleotides and NAD precursors and products in various biological samples.

Adenine nucleotides (AN) are ubiquitous metabolites that regulate cellular energy metabolism and modulate cell communication and inflammation. To understand how disturbances in AN balance arise and affect cellular function, robust quantification techniques for these metabolites are crucial. However, due to their hydrophilicity, simultaneous quantification of AN across various biological samples has been challenging. Here we present a hydrophilic interaction high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) based method for the quantification of 26 adenosine nucleotides and precursors as well as metabolic products of nicotinamide adenine dinucleotide (NAD) in plasma, liver, and adipose tissue samples as well as cell culture supernatants and cells. Method validation was performed with regard to linearity, accuracy, precision, matrix effects, and carryover. Finally, analysis of cell culture supernatants derived from intestinal organoids and RAW 264.7 cells illustrates that the here described method is a reliable and easy-to-use tool to quantify AN and opens up new avenues to understand the role of AN generation and breakdown for cellular functions.

HLA-DP on Epithelial Cells Enables Tissue Damage by NKp44+ Natural Killer Cells in Ulcerative Colitis.

BACKGROUND & AIMS: Ulcerative colitis (UC) is characterized by severe inflammation and destruction of the intestinal epithelium, and is associated with specific risk single nucleotide polymorphisms in HLA class II. Given the recently discovered interactions between subsets of HLA-DP molecules and the activating natural killer (NK) cell receptor NKp44, genetic associations of UC and HLA-DP haplotypes and their functional implications were investigated. METHODS: HLA-DP haplotype and UC risk association analyses were performed (UC: n = 13,927; control: n = 26,764). Expression levels of HLA-DP on intestinal epithelial cells (IECs) in individuals with and without UC were quantified. Human intestinal 3-dimensional (3D) organoid cocultures with human NK cells were used to determine functional consequences of interactions between HLA-DP and NKp44. RESULTS: These studies identified HLA-DPA1∗01:03-DPB1∗04:01 (HLA-DP401) as a risk haplotype and HLA-DPA1∗01:03-DPB1∗03:01 (HLA-DP301) as a protective haplotype for UC in European populations. HLA-DP expression was significantly higher on IECs of individuals with UC compared with controls. IECs in human intestinal 3D organoids derived from HLA-DP401pos individuals showed significantly stronger binding of NKp44 compared with HLA-DP301pos IECs. HLA-DP401pos IECs in organoids triggered increased degranulation and tumor necrosis factor production by NKp44+ NK cells in cocultures, resulting in enhanced epithelial cell death compared with HLA-DP301pos organoids. Blocking of HLA-DP401-NKp44 interactions (anti-NKp44) abrogated NK cell activity in cocultures. CONCLUSIONS: We identified an UC risk HLA-DP haplotype that engages NKp44 and activates NKp44+ NK cells, mediating damage to intestinal epithelial cells in an HLA-DP haplotype-dependent manner. The molecular interaction between NKp44 and HLA-DP401 in UC can be targeted by therapeutic interventions to reduce NKp44+ NK cell-mediated destruction of the intestinal epithelium in UC.

Human immunodeficiency virus and antiretroviral therapy-mediated immune cell metabolic dysregulation in children born to HIV-infected women: potential clinical implications.

Commencing lifelong antiretroviral therapy (ART) immediately following HIV diagnosis (Option B+) has dramatically improved the health of HIV-infected women and their children, with the majority being of HIV-exposed children born uninfected (HEU). This success has led to an increasing population of HIV-infected women receiving ART during pregnancy and children exposed to ART in utero. Nonetheless, a small proportion of children are still infected with HIV (HEI) each year. HEI children suffer from reduced immunocompetence and host-defence, due to CD4+ T lymphocyte depletion, but also dysregulation of other immune cells including CD8+ T lymphocytes, natural killer (NK) cells, macrophages including B lymphocytes. Furthermore, although HEU children are uninfected, altered immune responses are observed and associated with increased vulnerability to infections. The mechanisms underlying immune dysregulation in HEU children remain poorly described. Building on early studies, emerging data suggests that HIV/ART exposure early in life affects cell metabolic function of HEU children. Prenatal HIV/ART exposure has been associated with dysregulation of mitochondria, including impaired DNA polymerase activity. Furthermore, dysregulation of oxidative phosphorylation (OXPHOS) causes a decreased generation of adenosine triphosphate (ATP) and increased production of reactive oxygen species (ROS), resulting in oxidative stress. These altered metabolic processes can affect immune cell viability and immune responses. Recent studies have indicated that immune-metabolic dysregulation may contribute to HIV-associated pathogenesis and clinical observations associated with HIV and ART exposure in HEU/HEI children. Given the critical role metabolic processes in immune cell functioning, immune-metabolic dysregulation in HEU and HEI children may have implications in effective host-defence responses against pathogens, as well as efficacy of standard ART regimens and future novel HIV cure approaches in HEI children. At the same time, targeting metabolic pathways of immune cells may provide safer and novel approaches for HIV cure strategies. Here, we review the current literature investigating immune-metabolic dysregulation in paediatric HIV pathogenesis.

Expanded ILC2s in human infant intestines promote tissue growth.

Early life is characterized by extraordinary challenges, including rapid tissue growth and immune adaptation to foreign antigens after birth. During this developmental stage, infants have an increased risk of immune-mediated diseases. Here, we demonstrate that tissue-resident, interleukin (IL)-13- and IL-4-producing group 2 innate lymphoid cells (ILC2s) are enriched in human infant intestines compared to adult intestines. Organoid systems were employed to assess the role of infant intestinal ILC2s in intestinal development and showed that IL-13 and IL-4 increased epithelial cell proliferation and skewed cell differentiation toward secretory cells. IL-13 furthermore upregulated the production of mediators of type-2 immunity by infant intestinal epithelial cells, including vascular endothelial growth factor-A and IL-26, a chemoattractant for eosinophils. In line with these in vitro findings increased numbers of eosinophils were detected in vivo in infant intestines. Taken together, ILC2s are enriched in infant intestines and can support intestinal development while inducing an epithelial secretory response associated with type 2 immune-mediated diseases.

The co-inhibitory receptor TIGIT regulates NK cell function and is upregulated in human intrahepatic CD56bright NK cells.

The crosstalk between NK cells and their surrounding environment is enabled through activating and inhibitory receptors, which tightly control NK cell activity. The co-inhibitory receptor TIGIT decreases NK cell cytotoxicity and is involved in NK cell exhaustion, but has also been associated with liver regeneration, highlighting that the contribution of human intrahepatic CD56bright NK cells in regulating tissue homeostasis remains incompletely understood. A targeted single-cell mRNA analysis revealed distinct transcriptional differences between matched human peripheral blood and intrahepatic CD56bright NK cells. Multiparameter flow cytometry identified a cluster of intrahepatic NK cells with overlapping high expression of CD56, CD69, CXCR6, TIGIT and CD96. Intrahepatic CD56bright NK cells also expressed significantly higher protein surface levels of TIGIT, and significantly lower levels of DNAM-1 compared to matched peripheral blood CD56bright NK cells. TIGIT+ CD56bright NK cells showed diminished degranulation and TNF-α production following stimulation. Co-incubation of peripheral blood CD56bright NK cells with human hepatoma cells or primary human hepatocyte organoids resulted in migration of NK cells into hepatocyte organoids and upregulation of TIGIT and downregulation of DNAM-1 expression, in line with the phenotype of intrahepatic CD56bright NK cells. Intrahepatic CD56bright NK cells represent a transcriptionally, phenotypically, and functionally distinct population of NK cells that expresses higher levels of TIGIT and lower levels of DNAM-1 than matched peripheral blood CD56bright NK cells. Increased expression of inhibitory receptors by NK cells within the liver environment can contribute to tissue homeostasis and reduction of liver inflammation.

CXCR5+PD-1++ CD4+ T cells colonize infant intestines early in life and promote B cell maturation.

Gastrointestinal infections are a major cause for serious clinical complications in infants. The induction of antibody responses by B cells is critical for protective immunity against infections and requires CXCR5+PD-1++ CD4+ T cells (TFH cells). We investigated the ontogeny of CXCR5+PD-1++ CD4+ T cells in human intestines. While CXCR5+PD-1++ CD4+ T cells were absent in fetal intestines, CXCR5+PD-1++ CD4+ T cells increased after birth and were abundant in infant intestines, resulting in significant higher numbers compared to adults. These findings were supported by scRNAseq analyses, showing increased frequencies of CD4+ T cells with a TFH gene signature in infant intestines compared to blood. Co-cultures of autologous infant intestinal CXCR5+PD-1+/-CD4+ T cells with B cells further demonstrated that infant intestinal TFH cells were able to effectively promote class switching and antibody production by B cells. Taken together, we demonstrate that functional TFH cells are numerous in infant intestines, making them a promising target for oral pediatric vaccine strategies.

Enhanced Th17 responses in the appendix of children with complex compared to simple appendicitis are associated with microbial dysbiosis.

Introduction: Appendicitis is one of the most common causes of acute abdominal surgery in children. The clinical course of appendicitis ranges from simple to complex appendicitis. The mechanisms underlying the heterogeneity of appendicitis in children remain largely unclear. Dysregulated T cell responses play an important role in several inflammatory diseases of the intestine, but the extend of T cell dysregulation in appendicitis in children is less well known. Methods: To characterize appendiceal T cells in simple and complex appendicitis we performed in-depth immunophenotyping of appendiceal-derived T cells by flow cytometry and correlated this to appendiceal-derived microbiota analyses of the same patient. Results: Appendix samples of twenty children with appendicitis (n = 8 simple, n = 12 complex) were collected. T cells in complex appendicitis displayed an increased differentiated phenotype compared to simple appendicitis, including a loss of both CD27 and CD28 by CD4+ T cells and to a lesser extent by CD8+ T cells. Frequencies of phenotypic tissue-resident memory CD69+CD4+ T cells and CD69+CD8+ T cells were decreased in children with complex compared to simple appendicitis, indicating disruption of local tissue-resident immune responses. In line with the increased differentiated phenotype, cytokine production of in particular IL-17A by CD4+ T cells was increased in children with complex compared to simple appendicitis. Furthermore, frequencies of IL-17A+ CD4+ T cells correlated with a dysregulation of the appendiceal microbiota in children with complex appendicitis. Conclusion: In conclusion, disruption of local T cell responses, and enhanced pro-inflammatory Th17 responses correlating to changes in the appendiceal microbiota were observed in children with complex compared to simple appendicitis. Further studies are needed to decipher the role of a dysregulated network of microbiota and Th17 cells in the development of complex appendicitis in children.

KIR3DS1 directs NK cell-mediated protection against human adenovirus infections.

Human adenoviruses (HAdVs) are a major cause for disease in children, in particular after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Currently, effective therapies for HAdV infections in immunocompromised hosts are lacking. To decipher immune recognition of HAdV infection and determine new targets for immune-mediated control, we used an HAdV infection 3D organoid system, based on primary human intestinal epithelial cells. HLA-F, the functional ligand for the activating NK cell receptor KIR3DS1, was strongly up-regulated and enabled enhanced killing of HAdV5-infected cells in organoids by KIR3DS1+ NK cells. In contrast, HLA-A and HLA-B were significantly down-regulated in HAdV5-infected organoids in response to adenoviral E3/glycoprotein19K, consistent with evasion from CD8+ T cells. Immunogenetic analyses in a pediatric allo-HSCT cohort showed a reduced risk to develop severe HAdV disease and faster clearance of HAdV viremia in children receiving KIR3DS1+/HLA-Bw4+ donor cells compared with children receiving non­KIR3DS1+/HLA-Bw4+ cells. These findings identify the KIR3DS1/HLA-F axis as a new target for immunotherapeutic strategies against severe HAdV disease.

High Metabolic Function and Resilience of NKG2A-Educated NK Cells.

Natural killer (NK) cells are an important component of the innate immune system for the control of intracellular pathogens and cancer cells. NK cells demonstrate heterogeneous expression of inhibitory surface receptors. Signaling through these various receptors during NK cell development promotes functionality, referred to as NK cell education. Here we investigated the impact of education on NK cell metabolism through functional assessment of critical metabolic pathways and calcium signaling. Educated NK cells had an increased uptake of the metabolic substrates 2-NBDG, a fluorescent glucose analog, and BODIPY FL C16, a fluorescent palmitate, compared to uneducated NK cells. Comparison of NK cells educated via KIRs or NKG2A showed that NKG2A-educated NK cells were the main contributor to these differences in uptake of metabolites, and that NKG2A-educated NK cells were functionally more resilient in response to metabolic blockade of oxidative phosphorylation. Furthermore, NKG2A-educated NK cells exhibited higher peak calcium concentration following stimulation, indicating stronger signaling events taking place in these educated NK cells. These results demonstrate that cellular metabolism plays an important role in the functional differences observed between educated and uneducated NK cells, and show that NKG2A-educated NK cells remain more functionally competent than KIR-educated NK cells when oxidative phosphorylation is restricted. Understanding metabolic programming during NK cell education may unveil future targets to manipulate NK cell function for use in clinical settings, such as cancer therapies.

HIV-1 induced changes in HLA-C*03 :  04-presented peptide repertoires lead to reduced engagement of inhibitory natural killer cell receptors.

OBJECTIVE: Viral infections influence intracellular peptide repertoires available for presentation by HLA-I. Alterations in HLA-I/peptide complexes can modulate binding of killer immunoglobuline-like receptors (KIRs) and thereby the function of natural killer (NK) cells. Although multiple studies have provided evidence that HLA-I/KIR interactions play a role in HIV-1 disease progression, the consequence of HIV-1 infection for HLA-I/KIR interactions remain largely unknown. DESIGN: We determined changes in HLA-I presented peptides resulting from HIV-1-infection of primary human CD4 T cells and assessed the impact of changes in peptide repertoires on HLA-I/KIR interactions. METHODS: Liquid chromatography-coupled tandem mass spectrometry to identify HLA-I presented peptides, cell-based in-vitro assays to evaluate functional consequences of alterations in immunopeptidome and atomistic molecular dynamics simulations to confirm experimental data. RESULTS: A total of 583 peptides exclusively presented on HIV-1-infected cells were identified, of which only 0.2% represented HIV-1 derived peptides. Focusing on HLA-C*03 : 04/KIR2DL3 interactions, we observed that HLA-C*03 : 04-presented peptides derived from noninfected CD4 T cells mediated stronger binding of inhibitory KIR2DL3 than peptides derived from HIV-1-infected cells. Furthermore, the most abundant peptide presented by HLA-C*03 : 04 on noninfected CD4 T cells (VIYPARISL) mediated the strongest KIR2DL3-binding, while the most abundant peptide presented on HIV-1-infected cells (YAIQATETL) did not mediate KIR2DL3-binding. Molecular dynamics simulations of HLA-C*03 : 04/KIR2DL3 interactions in the context of these two peptides revealed that VIYPARISL significantly enhanced the HLA-C*03 : 04/peptide contact area to KIR2DL3 compared with YAIQATETL. CONCLUSION: These data demonstrate that HIV-1 infection-induced changes in HLA-I-presented peptides can reduce engagement of inhibitory KIRs, providing a mechanism for enhanced activation of NK cells by virus-infected cells.

The Transcription Factor Promyelocytic Leukemia Zinc Finger Protein Is Associated With Expression of Liver-Homing Receptors on Human Blood CD56bright Natural Killer Cells.

The transcription factor promyelocytic leukemia zinc finger protein (PLZF) is involved in the development of natural killer (NK) cells and innate lymphoid cells, including liver-resident NK cells in mice. In human NK cells, the role of PLZF in liver residency is still unknown. Expression of PLZF in matched human peripheral blood- and liver-derived NK cells and the association of PLZF expression with surface molecules and transcription factors relevant for tissue residency were investigated using multiparameter flow cytometry and assessing single-cell messenger RNA (mRNA) levels. Intrahepatic cluster of differentiation (CD)56bright NK cells expressed significantly higher levels of PLZF than peripheral blood CD56bright NK cells, which were predominantly PLZFlo. Expression of PLZF was highest within C-X-C motif chemokine receptor 6 (CXCR6)+CD69+ liver-resident NK cells among intrahepatic CD56bright NK cell populations. Association of PLZF with liver-residency markers was also reflected at mRNA levels. A small PLZFhiCD56bright NK cell population was identified in peripheral blood that also expressed the liver-residency markers CXCR6 and CD69 and shared functional characteristics with liver-resident NK cells. Conclusion: PLZF is implicated as part of a transcriptional network that promotes liver residency of human NK cells. Expression of liver-homing markers on peripheral blood PLZFhiCD56bright NK cells identifies an intermediate population potentially contributing to the maintenance of liver-resident NK cells.

Human Fetal TNF-α-Cytokine-Producing CD4+ Effector Memory T Cells Promote Intestinal Development and Mediate Inflammation Early in Life.

Although the fetal immune system is considered tolerogenic, preterm infants can suffer from severe intestinal inflammation, including necrotizing enterocolitis (NEC). Here, we demonstrate that human fetal intestines predominantly contain tumor necrosis factor-α (TNF-α)+CD4+CD69+ T effector memory (Tem) cells. Single-cell RNA sequencing of fetal intestinal CD4+ T cells showed a T helper 1 phenotype and expression of genes mediating epithelial growth and cell cycling. Organoid co-cultures revealed a dose-dependent, TNF-α-mediated effect of fetal intestinal CD4+ T cells on intestinal stem cell (ISC) development, in which low T cell numbers supported epithelial development, whereas high numbers abrogated ISC proliferation. CD4+ Tem cell frequencies were higher in inflamed intestines from preterm infants with NEC than in healthy infant intestines and showed enhanced TNF signaling. These findings reveal a distinct population of TNF-α-producing CD4+ T cells that promote mucosal development in fetal intestines but can also mediate inflammation upon preterm birth.