Mouse fetal growth restriction through parental and fetal immune gene variation and intercellular communications cascade.

Fetal growth restriction (FGR) affects 5-10% of pregnancies, and can have serious consequences for both mother and child. Prevention and treatment are limited because FGR pathogenesis is poorly understood. Genetic studies implicate KIR and HLA genes in FGR, however, linkage disequilibrium, genetic influence from both parents, and challenges with investigating human pregnancies make the risk alleles and their functional effects difficult to map. Here, we demonstrate that the interaction between the maternal KIR2DL1, expressed on uterine natural killer (NK) cells, and the paternally inherited HLA-C*0501, expressed on fetal trophoblast cells, leads to FGR in a humanized mouse model. We show that the KIR2DL1 and C*0501 interaction leads to pathogenic uterine arterial remodeling and modulation of uterine NK cell function. This initial effect cascades to altered transcriptional expression and intercellular communication at the maternal-fetal interface. These findings provide mechanistic insight into specific FGR risk alleles, and provide avenues of prevention and treatment.

Identification of early neurodegenerative pathways in progressive multiple sclerosis.

Progressive multiple sclerosis (MS) is characterized by unrelenting neurodegeneration, which causes cumulative disability and is refractory to current treatments. Drug development to prevent disease progression is an urgent clinical need yet is constrained by an incomplete understanding of its complex pathogenesis. Using spatial transcriptomics and proteomics on fresh-frozen human MS brain tissue, we identified multicellular mechanisms of progressive MS pathogenesis and traced their origin in relation to spatially distributed stages of neurodegeneration. By resolving ligand-receptor interactions in local microenvironments, we discovered defunct trophic and anti-inflammatory intercellular communications within areas of early neuronal decline. Proteins associated with neuronal damage in patient samples showed mechanistic concordance with published in vivo knockdown and central nervous system (CNS) disease models, supporting their causal role and value as potential therapeutic targets in progressive MS. Our findings provide a new framework for drug development strategies, rooted in an understanding of the complex cellular and signaling dynamics in human diseased tissue that facilitate this debilitating disease.

Identifying CNS-colonizing T cells as potential therapeutic targets to prevent progression of multiple sclerosis.

BACKGROUND: Multiple sclerosis (MS), an autoimmune disease of the central nervous system (CNS), can be suppressed in its early stages but eventually becomes clinically progressive and unresponsive to therapy. Here, we investigate whether the therapeutic resistance of progressive MS can be attributed to chronic immune cell accumulation behind the blood-brain barrier (BBB). METHODS: We systematically track CNS-homing immune cells in the peripheral blood of 31 MS patients and 31 matched healthy individuals in an integrated analysis of 497,705 single-cell transcriptomes and 355,433 surface protein profiles from 71 samples. Through spatial RNA sequencing, we localize these cells in post mortem brain tissue of 6 progressive MS patients contrasted against 4 control brains (20 samples, 85,000 spot transcriptomes). FINDINGS: We identify a specific pathogenic CD161+/lymphotoxin beta (LTB)+ T cell population that resides in brains of progressive MS patients. Intriguingly, our data suggest that the colonization of the CNS by these T cells may begin earlier in the disease course, as they can be mobilized to the blood by usage of the integrin-blocking antibody natalizumab in relapsing-remitting MS patients. CONCLUSIONS: As a consequence, we lay the groundwork for a therapeutic strategy to deplete CNS-homing T cells before they can fuel treatment-resistant progression. FUNDING: This study was supported by funding from the University Medical Center Hamburg-Eppendorf, the Stifterverband für die Deutsche Wissenschaft, the OAK Foundation, Medical Research Council UK, and Wellcome.

Structural and regulatory diversity shape HLA-C protein expression levels.

Expression of HLA-C varies widely across individuals in an allele-specific manner. This variation in expression can influence efficacy of the immune response, as shown for infectious and autoimmune diseases. MicroRNA binding partially influences differential HLA-C expression, but the additional contributing factors have remained undetermined. Here we use functional and structural analyses to demonstrate that HLA-C expression is modulated not just at the RNA level, but also at the protein level. Specifically, we show that variation in exons 2 and 3, which encode the α1/α2 domains, drives differential expression of HLA-C allomorphs at the cell surface by influencing the structure of the peptide-binding cleft and the diversity of peptides bound by the HLA-C molecules. Together with a phylogenetic analysis, these results highlight the diversity and long-term balancing selection of regulatory factors that modulate HLA-C expression.

Resolving TYK2 locus genotype-to-phenotype differences in autoimmunity.

Thousands of genetic variants have been identified, which contribute to the development of complex diseases, but determining how to elucidate their biological consequences for translation into clinical benefit is challenging. Conflicting evidence regarding the functional impact of genetic variants in the tyrosine kinase 2 (TYK2) gene, which is differentially associated with common autoimmune diseases, currently obscures the potential of TYK2 as a therapeutic target. We aimed to resolve this conflict by performing genetic meta-analysis across disorders; subsequent molecular, cellular, in vivo, and structural functional follow-up; and epidemiological studies. Our data revealed a protective homozygous effect that defined a signaling optimum between autoimmunity and immunodeficiency and identified TYK2 as a potential drug target for certain common autoimmune disorders.

Natural killer cells and their receptors in multiple sclerosis.

The immune system has crucial roles in the pathogenesis of multiple sclerosis. While the adaptive immune cell subsets, T and B cells, have been the main focus of immunological research in multiple sclerosis, it is now important to realize that the innate immune system also has a key involvement in regulating autoimmune responses in the central nervous system. Natural killer cells are innate lymphocytes that play vital roles in a diverse range of infections. There is evidence that they influence a number of autoimmune conditions. Recent studies in multiple sclerosis and its murine model, experimental autoimmune encephalomyelitis, are starting to provide some understanding of the role of natural killer cells in regulating inflammation in the central nervous system. Natural killer cells express a diverse range of polymorphic cell surface receptors, which interact with polymorphic ligands; this interaction controls the function and the activation status of the natural killer cell. In this review, we discuss evidence for the role of natural killer cells in multiple sclerosis and experimental autoimmune encephalomyelitis. We consider how a change in the balance of signals received by the natural killer cell influences its involvement in the ensuing immune response, in relation to multiple sclerosis.

TNF receptor 1 genetic risk mirrors outcome of anti-TNF therapy in multiple sclerosis.

Although there has been much success in identifying genetic variants associated with common diseases using genome-wide association studies (GWAS), it has been difficult to demonstrate which variants are causal and what role they have in disease. Moreover, the modest contribution that these variants make to disease risk has raised questions regarding their medical relevance. Here we have investigated a single nucleotide polymorphism (SNP) in the TNFRSF1A gene, that encodes tumour necrosis factor receptor 1 (TNFR1), which was discovered through GWAS to be associated with multiple sclerosis (MS), but not with other autoimmune conditions such as rheumatoid arthritis, psoriasis and Crohn's disease. By analysing MS GWAS data in conjunction with the 1000 Genomes Project data we provide genetic evidence that strongly implicates this SNP, rs1800693, as the causal variant in the TNFRSF1A region. We further substantiate this through functional studies showing that the MS risk allele directs expression of a novel, soluble form of TNFR1 that can block TNF. Importantly, TNF-blocking drugs can promote onset or exacerbation of MS, but they have proven highly efficacious in the treatment of autoimmune diseases for which there is no association with rs1800693. This indicates that the clinical experience with these drugs parallels the disease association of rs1800693, and that the MS-associated TNFR1 variant mimics the effect of TNF-blocking drugs. Hence, our study demonstrates that clinical practice can be informed by comparing GWAS across common autoimmune diseases and by investigating the functional consequences of the disease-associated genetic variation.

Characterisation of Foxp3 splice variants in human CD4+ and CD8+ T cells--identification of Foxp3Δ7 in human regulatory T cells.

Foxp3 is proposed to play a critical role in the development and function of regulatory T cells. Functional and transgenic studies in mice propose Foxp3 as a "regulatory T cell lineage specification factor" but conflicting data exist in humans. Expression of multiple Foxp3 splice variants in humans represents an additional layer of complexity for this transcription factor and acts as a possible mechanism of regulating protein diversity. We report the identification of a novel splice variant of Foxp3, called Foxp3Δ7, in ex vivo CD4+CD25+ T cells and CD8+ regulatory T cell clones. Foxp3Δ7 lacks the 81bp region that encodes exon 7 of Foxp3, which is a part of the leucine zipper domain of the protein. The three splice variants of Foxp3 namely Foxp3FL, Foxp3Δ2 and Foxp3Δ7 are co-expressed in ex vivo human CD4+CD25+ T cells and CD8+ Treg clones. Stimulation of freshly isolated CD4+CD25+ T cells with anti-CD3 and anti-CD28 antibodies leads to a 140-fold upregulation of Foxp3Δ7 within 24h of stimulation, which is ∼10-fold greater than that observed in stimulated CD4+CD25- T cells. In addition, resting CD8+ Treg cells have decreased expression of Foxp3FL and Foxp3Δ2; however they have a 10-fold higher expression of Foxp3Δ7, in comparison to ex vivo CD4+CD25+ T cells. In order to assess the functional effects of these Foxp3 isoforms, we carried out lentivirus expression studies. All three isoforms were capable of inducing increased levels of CD25 expression in primary human CD4+ T cells, along with a tendency to decreased levels of CD127. Further investigation into pathways that alter the relative proportions of Foxp3 isoforms, and hence their interaction with other transcriptional co-regulators, will help to define the role of Foxp3 isoforms in immune regulation.

A role of cellular prion protein in programming T-cell cytokine responses in disease.

The cellular prion protein (PrP(C)) is widely expressed in neural and non-neural tissues, but its function is unknown. Elucidation of the part played by PrP(C) in adaptive immunity has been a particular conundrum: increased expression of cell surface PrP(C) has been documented during T-cell activation, yet the functional significance of this activation remains unclear, with conflicting data on the effects of Prnp gene knockout on various parameters of T-cell immunity. We show here that Prnp mRNA is highly inducible within 8-24 h of T-cell activation, with surface protein levels rising from 24 h. When measured in parallel with CD69 and CD25, PrP(C) is a late activation antigen. Consistent with its up-regulation being a late activation event, PrP deletion did not alter T-cell-antigen presenting cell conjugate formation. Most important, activated PrP(0/0) T cells demonstrated much reduced induction of several T helper (Th) 1, Th2, and Th17 cytokines, whereas others, such as TNF-alpha and IL-9, were unaffected. These changes were investigated in the context of an autoimmune model and a bacterial challenge model. In experimental autoimmune encephalomyelitis, PrP-knockout mice showed enhanced disease in the face of reduced IL-17 responses. In a streptococcal sepsis model, this constrained cytokine program was associated with poorer local control of infection, although with reduced bacteremia. The findings indicate that PrP(C) is a potentially important molecule influencing T-cell activation and effector function.

The cellular prion protein is preferentially expressed by CD4+ CD25+ Foxp3+ regulatory T cells.

Post-translational modification of the cellular prion protein (PrP(C)) is intimately associated with the pathogenesis of prion disease, yet the normal function of the protein remains unclear. PrP(C) is expressed in lymphoid cells and is known to be a T-cell activation antigen. Further, transcription profiling studies of regulatory T cells have shown preferential overexpression of PrP(C), suggesting a possible role in regulatory function. We report that both the expression of PrP message and cell surface PrP(C) levels are increased in murine CD4(+) CD25(+) regulatory T cells compared with CD4(+) CD25(-) cells. However, PrP(0/0) mice do not show altered regulatory T-cell numbers or forkhead box P3 (Foxp3) expression levels, or impaired regulatory T-cell function in vitro. Nevertheless, the preferential expression of surface PrP(C) by regulatory T cells raises the possibility that therapeutic ligation of PrP(C) might alter immune regulation.

CD27 expression discriminates between regulatory and non-regulatory cells after expansion of human peripheral blood CD4+ CD25+ cells.

It is clear that regulatory T cells (Treg) have an important role in preventing autoimmunity and modulating responses to pathogens. Full characterization of Treg cell function in human patients would be greatly facilitated by practical methods for expanding Treg in vitro. Methods for expansion have been reported but whether expression of surface and intracellular markers associated with freshly isolated Treg following expansion correlates with the maintenance of function is unclear. Our aim was to investigate the various methods of expansion and to correlate regulatory activity with expression of these markers. We show that, of the markers associated with freshly isolated Treg, only CD27 expression correlated with regulatory activity and could be used to isolate cells with regulatory activity from lines expanded from CD4+ CD25+ cells. Also, cells expressing high levels of the transcription factor forkhead box P3 (Foxp3) were confined to the CD27+ population within these lines. Expression of CD27 by cells in lines expanded from CD4+ CD25- cells varied depending on the stimulus used for expansion, but these lines did not have significant regulatory activity even when the CD27+ cells were tested. Analysis of synovial CD4+ CD25+ cells from reactive arthritis patients revealed that they were predominantly CD27 positive. This also applied to CD25(high) and CD25(intermediate) CD4+ cells, despite their reported different abilities to regulate. We conclude that, whilst CD27 is useful for identifying Treg in the cell lines obtained after expansion of CD4+ CD25+ cells, its expression may not reliably identify the Treg cell population in other T-cell populations such as those found in joints.