NKp44/HLA-DP-dependent regulation of CD8 effector T cells by NK cells.

Although natural killer (NK) cells are recognized for their modulation of immune responses, the mechanisms by which human NK cells mediate immune regulation are unclear. Here, we report that expression of human leukocyte antigen (HLA)-DP, a ligand for the activating NK cell receptor NKp44, is significantly upregulated on CD8+ effector T cells, in particular in human cytomegalovirus (HCMV)+ individuals. HLA-DP+ CD8+ T cells expressing NKp44-binding HLA-DP antigens activate NKp44+ NK cells, while HLA-DP+ CD8+ T cells not expressing NKp44-binding HLA-DP antigens do not. In line with this, frequencies of HLA-DP+ CD8+ T cells are increased in individuals not encoding for NKp44-binding HLA-DP haplotypes, and contain hyper-expanded CD8+ T cell clones, compared to individuals expressing NKp44-binding HLA-DP molecules. These findings identify a molecular interaction facilitating the HLA-DP haplotype-specific editing of HLA-DP+ CD8+ T cell effector populations by NKp44+ NK cells and preventing the generation of hyper-expanded T cell clones, which have been suggested to have increased potential for autoimmunity.

HLA-DPA1*02:01~B1*01:01 is a risk haplotype for primary sclerosing cholangitis mediating activation of NKp44+ NK cells.

OBJECTIVE: Primary sclerosing cholangitis (PSC) is characterised by bile duct strictures and progressive liver disease, eventually requiring liver transplantation. Although the pathogenesis of PSC remains incompletely understood, strong associations with HLA-class II haplotypes have been described. As specific HLA-DP molecules can bind the activating NK-cell receptor NKp44, we investigated the role of HLA-DP/NKp44-interactions in PSC. DESIGN: Liver tissue, intrahepatic and peripheral blood lymphocytes of individuals with PSC and control individuals were characterised using flow cytometry, immunohistochemical and immunofluorescence analyses. HLA-DPA1 and HLA-DPB1 imputation and association analyses were performed in 3408 individuals with PSC and 34 213 controls. NK cell activation on NKp44/HLA-DP interactions was assessed in vitro using plate-bound HLA-DP molecules and HLA-DPB wildtype versus knock-out human cholangiocyte organoids. RESULTS: NKp44+NK cells were enriched in livers, and intrahepatic bile ducts of individuals with PSC showed higher expression of HLA-DP. HLA-DP haplotype analysis revealed a highly elevated PSC risk for HLA-DPA1*02:01~B1*01:01 (OR 1.99, p=6.7×10-50). Primary NKp44+NK cells exhibited significantly higher degranulation in response to plate-bound HLA-DPA1*02:01-DPB1*01:01 compared with control HLA-DP molecules, which were inhibited by anti-NKp44-blocking. Human cholangiocyte organoids expressing HLA-DPA1*02:01-DPB1*01:01 after IFN-γ-exposure demonstrated significantly increased binding to NKp44-Fc constructs compared with unstimulated controls. Importantly, HLA-DPA1*02:01-DPB1*01:01-expressing organoids increased degranulation of NKp44+NK cells compared with HLA-DPB1-KO organoids. CONCLUSION: Our studies identify a novel PSC risk haplotype HLA-DP A1*02:01~DPB1*01:01 and provide clinical and functional data implicating NKp44+NK cells that recognise HLA-DPA1*02:01-DPB1*01:01 expressed on cholangiocytes in PSC pathogenesis.

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.

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.

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.

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.

In vitro co-culture of human intestinal organoids and lamina propria-derived CD4+ T cells.

Crosstalk between immune cells and intestinal stem cells (ISCs) in vivo plays a critical role in tissue homeostasis and inflammation; however, in vitro models based on primary cells recapitulating this interaction were lacking. Here, we provide a detailed protocol for an autologous in vitro long-term 3D co-culture system of human intestinal CD4+ T cells and ISCs to study T cell-intestinal epithelial cell interactions during tissue development and inflammation. For complete details on the use and execution of this protocol, please refer to Schreurs et al. (2019).

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.