Human Epistatic Interaction Controls IL7R Splicing and Increases Multiple Sclerosis Risk.

Multiple sclerosis (MS) is an autoimmune disorder where T cells attack neurons in the central nervous system (CNS) leading to demyelination and neurological deficits. A driver of increased MS risk is the soluble form of the interleukin-7 receptor alpha chain gene (sIL7R) produced by alternative splicing of IL7R exon 6. Here, we identified the RNA helicase DDX39B as a potent activator of this exon and consequently a repressor of sIL7R, and we found strong genetic association of DDX39B with MS risk. Indeed, we showed that a genetic variant in the 5' UTR of DDX39B reduces translation of DDX39B mRNAs and increases MS risk. Importantly, this DDX39B variant showed strong genetic and functional epistasis with allelic variants in IL7R exon 6. This study establishes the occurrence of biological epistasis in humans and provides mechanistic insight into the regulation of IL7R exon 6 splicing and its impact on MS risk.

Spatial map of human T cell compartmentalization and maintenance over decades of life.

Mechanisms for human memory T cell differentiation and maintenance have largely been inferred from studies of peripheral blood, though the majority of T cells are found in lymphoid and mucosal sites. We present here a multidimensional, quantitative analysis of human T cell compartmentalization and maintenance over six decades of life in blood, lymphoid, and mucosal tissues obtained from 56 individual organ donors. Our results reveal that the distribution and tissue residence of naive, central, and effector memory, and terminal effector subsets is contingent on both their differentiation state and tissue localization. Moreover, T cell homeostasis driven by cytokine or TCR-mediated signals is different in CD4+ or CD8+ T cell lineages, varies with their differentiation stage and tissue localization, and cannot be inferred from blood. Our data provide an unprecedented spatial and temporal map of human T cell compartmentalization and maintenance, supporting distinct pathways for human T cell fate determination and homeostasis.

The heterogeneity of human CD127(+) innate lymphoid cells revealed by single-cell RNA sequencing.

Innate lymphoid cells (ILCs) are increasingly appreciated as important participants in homeostasis and inflammation. Substantial plasticity and heterogeneity among ILC populations have been reported. Here we have delineated the heterogeneity of human ILCs through single-cell RNA sequencing of several hundreds of individual tonsil CD127(+) ILCs and natural killer (NK) cells. Unbiased transcriptional clustering revealed four distinct populations, corresponding to ILC1 cells, ILC2 cells, ILC3 cells and NK cells, with their respective transcriptomes recapitulating known as well as unknown transcriptional profiles. The single-cell resolution additionally divulged three transcriptionally and functionally diverse subpopulations of ILC3 cells. Our systematic comparison of single-cell transcriptional variation within and between ILC populations provides new insight into ILC biology during homeostasis, with additional implications for dysregulation of the immune system.

Large-Scale HLA Tetramer Tracking of T Cells during Dengue Infection Reveals Broad Acute Activation and Differentiation into Two Memory Cell Fates.

T cells play important multifaceted roles during dengue infection, and understanding their responses is important for defining correlates of protective immunity and identifying effective vaccine antigens. Using mass cytometry and a highly multiplexed peptide-HLA (human leukocyte antigen) tetramer staining strategy, we probed T cells from dengue patients-a total of 430 dengue and control candidate epitopes-together with key markers of activation, trafficking, and differentiation. During acute disease, dengue-specific CD8+ T cells expressed a distinct profile of activation and trafficking receptors that distinguished them from non-dengue-specific T cells. During convalescence, dengue-specific T cells differentiated into two major cell fates, CD57+ CD127--resembling terminally differentiated senescent memory cells and CD127+ CD57--resembling proliferation-capable memory cells. Validation in an independent cohort showed that these subsets remained at elevated frequencies up to one year after infection. These analyses aid our understanding of the generation of T cell memory in dengue infection or vaccination.

Signature of long-lived memory CD8+ T cells in acute SARS-CoV-2 infection.

Immunological memory is a hallmark of adaptive immunity and facilitates an accelerated and enhanced immune response upon re-infection with the same pathogen1,2. Since the outbreak of the ongoing COVID-19 pandemic, a key question has focused on which SARS-CoV-2-specific T cells stimulated during acute infection give rise to long-lived memory T cells3. Here, using spectral flow cytometry combined with cellular indexing of transcriptomes and T cell receptor sequencing, we longitudinally characterized individual SARS-CoV-2-specific CD8+ T cells of patients with COVID-19 from acute infection to 1 year into recovery and found a distinct signature identifying long-lived memory CD8+ T cells. SARS-CoV-2-specific memory CD8+ T cells persisting 1 year after acute infection express CD45RA, IL-7 receptor-α and T cell factor 1, but they maintain low expression of CCR7, thus resembling CD45RA+ effector memory T cells. Tracking individual clones of SARS-CoV-2-specific CD8+ T cells, we reveal that an interferon signature marks clones that give rise to long-lived cells, whereas prolonged proliferation and mechanistic target of rapamycin signalling are associated with clonal disappearance from the blood. Collectively, we describe a transcriptional signature that marks long-lived, circulating human memory CD8+ T cells following an acute viral infection.

Runx3 specifies lineage commitment of innate lymphoid cells.

Subsets of innate lymphoid cells (ILCs) reside in the mucosa and regulate immune responses to external pathogens. While ILCs can be phenotypically classified into ILC1, ILC2 and ILC3 subsets, the transcriptional control of commitment to each ILC lineage is incompletely understood. Here we report that the transcription factor Runx3 was essential for the normal development of ILC1 and ILC3 cells but not of ILC2 cells. Runx3 controlled the survival of ILC1 cells but not of ILC3 cells. Runx3 was required for expression of the transcription factor RORγt and its downstream target, the transcription factor AHR, in ILC3 cells. The absence of Runx3 in ILCs exacerbated infection with Citrobacter rodentium. Therefore, our data establish Runx3 as a key transcription factor in the lineage-specific differentiation of ILC1 and ILC3 cells.

Two waves of distinct hematopoietic progenitor cells colonize the fetal thymus.

The generation of T cells depends on the migration of hematopoietic progenitor cells to the thymus throughout life. The identity of the thymus-settling progenitor cells has been a matter of considerable debate. Here we found that thymopoiesis was initiated by a first wave of T cell lineage-restricted progenitor cells with limited capacity for population expansion but accelerated differentiation into mature T cells. They gave rise to αß and γδ T cells that constituted Vγ3(+) dendritic epithelial T cells. Thymopoiesis was subsequently maintained by less-differentiated progenitor cells that retained the potential to develop into B cells and myeloid cells. In that second wave, which started before birth, progenitor cells had high proliferative capacity but delayed differentiation capacity and no longer gave rise to embryonic γδ T cells. Our work reconciles conflicting hypotheses on the nature of thymus-settling progenitor cells.

The IL-7R antagonist lusvertikimab reduces leukemic burden in xenograft ALL via antibody-dependent cellular phagocytosis.

ABSTRACT: Acute lymphoblastic leukemia (ALL) arises from the uncontrolled proliferation of B-cell precursors (BCP-ALL) or T cells (T-ALL). Current treatment protocols obtain high cure rates in children but are based on toxic polychemotherapy. Novel therapies are urgently needed, especially in relapsed/refractory (R/R) disease, high-risk (HR) leukemias and T-ALL, in which immunotherapy approaches remain scarce. Although the interleukin-7 receptor (IL-7R) plays a pivotal role in ALL development, no IL-7R-targeting immunotherapy has yet reached clinical application in ALL. The IL-7Rα chain (CD127)-targeting IgG4 antibody lusvertikimab (LUSV; formerly OSE-127) is a full antagonist of the IL-7R pathway, showing a good safety profile in healthy volunteers. Here, we show that ∼85% of ALL cases express surface CD127. We demonstrate significant in vivo efficacy of LUSV immunotherapy in a heterogeneous cohort of BCP- and T-ALL patient-derived xenografts (PDX) in minimal residual disease (MRD) and overt leukemia models, including R/R and HR leukemias. Importantly, LUSV was particularly effective when combined with polychemotherapy in a phase 2-like PDX study with CD127high samples leading to MRD-negativity in >50% of mice treated with combination therapy. Mechanistically, LUSV targeted ALL cells via a dual mode of action comprising direct IL-7R antagonistic activity and induction of macrophage-mediated antibody-dependent cellular phagocytosis (ADCP). LUSV-mediated in vitro ADCP levels significantly correlated with CD127 expression levels and the reduction of leukemia burden upon treatment of PDX animals in vivo. Altogether, through its dual mode of action and good safety profile, LUSV may represent a novel immunotherapy option for any CD127+ ALL, particularly in combination with standard-of-care polychemotherapy.

Molecular and Functional Characterization of Lymphoid Progenitor Subsets Reveals a Bipartite Architecture of Human Lymphopoiesis.

The classical model of hematopoiesis established in the mouse postulates that lymphoid cells originate from a founder population of common lymphoid progenitors. Here, using a modeling approach in humanized mice, we showed that human lymphoid development stemmed from distinct populations of CD127- and CD127+ early lymphoid progenitors (ELPs). Combining molecular analyses with in vitro and in vivo functional assays, we demonstrated that CD127- and CD127+ ELPs emerged independently from lympho-mono-dendritic progenitors, responded differently to Notch1 signals, underwent divergent modes of lineage restriction, and displayed both common and specific differentiation potentials. Whereas CD127- ELPs comprised precursors of T cells, marginal zone B cells, and natural killer (NK) and innate lymphoid cells (ILCs), CD127+ ELPs supported production of all NK cell, ILC, and B cell populations but lacked T potential. On the basis of these results, we propose a "two-family" model of human lymphoid development that differs from the prevailing model of hematopoiesis.

Novel Synonymous Variant in IL7R Causes Preferential Expression of the Soluble Isoform.

PURPOSE: The interleukin-7 receptor (IL-7R) is primarily expressed on lymphoid cells and plays a crucial role in the development, proliferation, and survival of T cells. Autosomal recessive mutations that disrupt IL-7Rα chain expression give rise to a severe combined immunodeficiency (SCID), which is characterized by lymphopenia and a T-B+NK+ phenotype. The objective here was to diagnose two siblings displaying the T-B+NK+ SCID phenotype as initial clinical genetic testing did not detect any variants in known SCID genes. METHODS: Whole genome sequencing (WGS) was utilized to identify potential variants causing the SCID phenotype. Splicing prediction tools were employed to assess the deleterious impact of the mutation. Polymerase Chain Reaction (PCR), Sanger sequencing, flow cytometry, and ELISA were then used to validate the pathogenicity of the detected mutation. RESULTS: We discovered a novel homozygous synonymous mutation in the IL7R gene. Our functional studies indicate that this variant is pathogenic, causing exon 6, which encodes the transmembrane domain, to be preferentially spliced out. CONCLUSION: In this study, we identified a novel rare synonymous mutation causing a loss of IL-7Rα expression at the cellular membrane. This case demonstrates the value of reanalyzing genetic data based on the clinical phenotype and highlights the significance of functional studies in determining the pathogenicity of genetic variants.

Interleukin-12 and -23 Control Plasticity of CD127(+) Group 1 and Group 3 Innate Lymphoid Cells in the Intestinal Lamina Propria.

Human group 1 ILCs consist of at least three phenotypically distinct subsets, including NK cells, CD127(+) ILC1, and intraepithelial CD103(+) ILC1. In inflamed intestinal tissues from Crohn's disease patients, numbers of CD127(+) ILC1 increased at the cost of ILC3. Here we found that differentiation of ILC3 to CD127(+) ILC1 is reversible in vitro and in vivo. CD127(+) ILC1 differentiated to ILC3 in the presence of interleukin-2 (IL-2), IL-23, and IL-1ß dependent on the transcription factor RORγt, and this process was enhanced in the presence of retinoic acid. Furthermore, we observed in resection specimen from Crohn's disease patients a higher proportion of CD14(+) dendritic cells (DC), which in vitro promoted polarization from ILC3 to CD127(+) ILC1. In contrast, CD14(-) DCs promoted differentiation from CD127(+) ILC1 toward ILC3. These observations suggest that environmental cues determine the composition, function, and phenotype of CD127(+) ILC1 and ILC3 in the gut.

iNKT subsets differ in their developmental and functional requirements on Foxo1.

Invariant natural killer T (iNKT) cells play important roles in regulating immune responses. Based on cytokine profiling and key transcriptional factors, iNKT cells are classified into iNKT1, iNKT2, and iNKT17 subsets. However, whether the development and functions of these subsets are controlled by distinct mechanisms remains unclear. Here, we show that forkhead box protein O1 (Foxo1) promotes differentiation of iNKT1 and iNKT2 cells but not iNKT17 cells because of its distinct contributions to IL7R expression in these subsets. Nuclear Foxo1 is essential for Il7r expression in iNKT1 and iNKT2 cells at early stages of differentiation but is dispensable in iNKT17 cells. RORγt, instead of Foxo1, promotes IL7R expression in iNKT17 cells. Additionally, Foxo1 is required for the effector function of iNKT1 and iNKT2 cells but not iNKT17 cells. Cytoplasmic Foxo1 promotes activation of mTORC1 in iNKT1 and iNKT2 cells through inhibiting TSC1-TSC2 interaction, whereas it is dispensable for mTORC1 activation in iNKT17 cells. iNKT17 cells display distinct metabolic gene expression patterns from iNKT1 and iNKT2 cells that match their different functional requirements on Foxo1. Together, our results demonstrate that iNKT cell subsets differ in their developmental and functional requirements on Foxo1.

Cytokine-induced transient monocyte IL-7Ra expression and the serum milieu in tuberculosis.

Bacterial components and cytokines induce IL-7 receptor (IL-7Rα) expression in monocytes. Aberrant low IL-7Rα expression of monocytes has been identified as a feature of tuberculosis immunopathology. Here, we investigated the mechanisms underlying IL-7Rα regulation of monocytes and tuberculosis serum effects on IL-7Rα expression. Serum samples from tuberculosis patients and healthy controls, cytokine candidates, and mycobacterial components were analyzed for in vitro effects on IL-7Rα expression of primary monocytes, monocyte-derived macrophages (MDM), and monocyte cell lines. IL-7Rα regulation during culture and the role of FoxO1 were characterized. In vitro activation-induced IL-7Rα expression in human monocytes and serum samples from tuberculosis patients boosted IL-7Rα expression. Although pathognomonic tuberculosis cytokines were not associated with serum effects, we identified cytokines (i.e., GM-CSF, IL-1ß, TNF-α, IFN-γ) that induced IL-7Rα expression in monocytes and/or MDM comparable to mycobacterial components. Blocking of cytokine subsets (i.e., IL-1ß/TNF-α in monocytes, GM-CSF in MDM) largely diminished IL-7Rα expression induced by mycobacterial components. Finally, we showed that in vitro-induced IL-7Rα expression was transient and dependent on constitutive FoxO1 expression in primary monocytes and monocyte cell lines. This study demonstrated the crucial roles of cytokines and constitutive FoxO1 expression for transient IL-7Rα expression in monocytes.

IL7RA rs10491434 polymorphism is related to spontaneous HIV infection control in naïve HIV-infected patients: A retrospective study.

Interleukin 7 receptor (IL7R) is vital in the adaptive immune response against human immunodeficiency viruses (HIV). We assessed IL7RA polymorphisms (SNPs) in antiretroviral therapy (ART)-naïve HIV patients for their association with spontaneous HIV infection control. We conducted a retrospective cohort study involving 667 ART-naïve patients categorized by HIV progression (ordinal variable): 150 rapid progressors, 334 moderate/typical progressors, 86 long-term nonprogressors elite controllers (LTNPs-EC), and 97 LTNPs-non-EC. We genotyped three IL7RA SNPs using Agena Bioscience's MassARRAY platform. The association between IL7RA SNPs and spontaneous HIV infection control was evaluated using ordinal logistic regression. Individuals carrying the rs10491434 G allele have a higher likelihood of spontaneous HIV infection control (adjusted odds ratio [aOR] = 1.33; p = 0.023). Moreover, the IL7RA GCT haplotype, consisting of three specific SNPs (rs6897932, rs987106, and rs10491434), demonstrated an association with the control of untreated HIV infection (aOR = 1.34; p = 0.050). Remarkably, the rs10491434 SNP and the IL7RA GCT haplotype exhibited similar aOR values, suggesting that rs10491434 may be primarily responsible for the observed effect of the haplotype. IL7RA rs10491434 G allele is associated with a higher likelihood of spontaneous HIV infection control, indicating its significant role in the pathogenesis of HIV, possibly influencing infection course and viral replication control.

Interleukin-7 regulates CD127 expression and promotes CD8+ T cell activity in patients with primary cutaneous melanoma.

BACKGROUND: Interleukin (IL)-7 signaling through CD127 is impaired in lymphocytes in cancers and chronic infections, resulting in CD8+ T cell exhaustion. The mechanisms underlying CD8+ T cell responses to IL-7 in melanoma remain not completely elucidated. We previously showed reduced IL-7 level in melanoma patients. Thus, the aim of this study was to investigate the effect of IL-7 regulation on CD127 expression and CD8+ T cell responses in melanoma. METHODS: Healthy controls and primary cutaneous melanoma patients were enrolled. Membrane-bound CD127 (mCD127) expression on CD8+ T cells was determined by flow cytometry. Soluble CD127 (sCD127) protein level was measured by ELISA. Total CD127 and sCD127 mRNA level was measured by real-time PCR. CD8+ T cells were stimulated with recombinant human IL-7, along with signaling pathway inhibitors. CD8+ T cells were co-cultured with melanoma cell line, and the cytotoxicity of CD8+ T cells was assessed by measurement of lactate dehydrogenase expression. RESULTS: Plasma sCD127 was lower in melanoma patients compared with controls. The percentage of CD8+ T cells expressing mCD127 was higher, while sCD127 mRNA level was lower in peripheral and tumor-infiltrating CD8+ T cells from melanoma patients. There was no significant difference of total CD127 mRNA expression in CD8+ T cells between groups. IL-7 stimulation enhanced total CD127 and sCD127 mRNA expression and sCD127 release by CD8+ T cells. However, mCD127 mRNA expression on CD8+ T cells was not affected. This process was mainly mediated by phosphatidylinositol 3-kinase (PI3K) pathway. CD8+ T cells from melanoma patients exhibited decreased cytotoxicity. IL-7 stimulation promoted CD8+ T cell cytotoxicity, while inhibition of PI3K dampened IL-7-induced elevation of CD8+ T cell cytotoxicity. CONCLUSION: The current data suggested that insufficient IL-7 secretion might contribute to CD8+ T cell exhaustion and CD127 dysregulation in patients with primary cutaneous melanoma.

Interleukin-7-dependent nonclassical monocytes and CD40 expression are affected in children with type 1 diabetes.

The important role of IL-7 in the generation of self-reactive T-cells in autoimmune diseases is well established. Recent studies on autoimmunity-associated genetic polymorphisms indicated that differential IL-7 receptor (IL-7R) expression of monocytes may play a role in the underlying pathogenesis. The relevance of IL-7-mediated monocyte functions in type 1 diabetes remains elusive. In the present study, we characterized monocyte phenotype and IL-7-mediated effects in children with type 1 diabetes and healthy controls with multicolor flow cytometry and t-distributed Stochastic Neighbor-Embedded (t-SNE)-analyses. IL-7R expression of monocytes rapidly increased in vitro and was boosted through LPS. In the presence of IL-7, we detected lower monocyte IL-7R expression in type 1 diabetes patients as compared to healthy controls. This difference was most evident for the subset of nonclassical monocytes, which increased after IL-7 stimulation. t-SNE analyses revealed IL-7-dependent differences in monocyte subset distribution and expression of activation and maturation markers (i.e., HLA-DR, CD80, CD86, CD40). Notably, monocyte CD40 expression increased considerably by IL-7 and CD40/IL-7R co-expression differed between patients and controls. This study shows the unique effects of IL-7 on monocyte phenotype and functions. Lower IL-7R expression on IL-7-induced CD40high monocytes and impaired IL-7 response characterize monocytes from patients with type 1 diabetes.

Identification of human cytotoxic ILC3s.

Human ILCs are classically categorized into five subsets; cytotoxic CD127- CD94+ NK cells and non-cytotoxic CD127+ CD94- , ILC1s, ILC2s, ILC3s, and LTi cells. Here, we identify a previously unrecognized subset within the CD127+ ILC population, characterized by the expression of the cytotoxic marker CD94. These CD94+ ILCs resemble conventional ILC3s in terms of phenotype, transcriptome, and cytokine production, but are highly cytotoxic. IL-15 was unable to induce differentiation of CD94+ ILCs toward mature NK cells. Instead, CD94+ ILCs retained RORγt, CD127 and CD200R1 expression and produced IL-22 in response to IL-15. Culturing non-cytotoxic ILC3s with IL-12 induced upregulation of CD94 and cytotoxic activity, effects that were not observed with IL-15 stimulation. Thus, human helper ILCs can acquire a cytotoxic program without differentiating into NK cells.

Functional characterisation of two receptor interaction determinants in human thymic stromal lymphopoietin.

Thymic stromal lymphopoietin (TSLP) is a pro-inflammatory cytokine with important pathological roles in Asthma bronchiale, malignant tumours and other diseases. The heterodimeric human TSLP receptor (hTSLPR) consists of the TSLP-binding subunit (TSLPRα) and the IL-7Rα-subunit. We studied the properties of hTSLP variants with mutations in their bipartite interaction interface towards IL-7Rα. One mutant (T46D/K101D) showed only mild impairment in receptor affinity but a massive reduction in biological activity. To facilitate the future development of hTSLP mutants with drug properties, we have devised a eukaryontic cytokine display assay with activity read-out and intrinsic genotype-phenotype coupling.

Tissue memory CD4+ T cells expressing IL-7 receptor-alpha (CD127) preferentially support latent HIV-1 infection.

The primary reservoir for HIV is within memory CD4+ T cells residing within tissues, yet the features that make some of these cells more susceptible than others to infection by HIV is not well understood. Recent studies demonstrated that CCR5-tropic HIV-1 efficiently enters tissue-derived memory CD4+ T cells expressing CD127, the alpha chain of the IL7 receptor, but rarely completes the replication cycle. We now demonstrate that the inability of HIV to replicate in these CD127-expressing cells is not due to post-entry restriction by SAMHD1. Rather, relative to other memory T cell subsets, these cells are highly prone to undergoing latent infection with HIV, as revealed by the high levels of integrated HIV DNA in these cells. Host gene expression profiling revealed that CD127-expressing memory CD4+ T cells are phenotypically distinct from other tissue memory CD4+ T cells, and are defined by a quiescent state with diminished NFκB, NFAT, and Ox40 signaling. However, latently-infected CD127+ cells harbored unspliced HIV transcripts and stimulation of these cells with anti-CD3/CD28 reversed latency. These findings identify a novel subset of memory CD4+ T cells found in tissue and not in blood that are preferentially targeted for latent infection by HIV, and may serve as an important reservoir to target for HIV eradication efforts.