High expression of oleoyl-ACP hydrolase underpins life-threatening respiratory viral diseases.

Respiratory infections cause significant morbidity and mortality, yet it is unclear why some individuals succumb to severe disease. In patients hospitalized with avian A(H7N9) influenza, we investigated early drivers underpinning fatal disease. Transcriptomics strongly linked oleoyl-acyl-carrier-protein (ACP) hydrolase (OLAH), an enzyme mediating fatty acid production, with fatal A(H7N9) early after hospital admission, persisting until death. Recovered patients had low OLAH expression throughout hospitalization. High OLAH levels were also detected in patients hospitalized with life-threatening seasonal influenza, COVID-19, respiratory syncytial virus (RSV), and multisystem inflammatory syndrome in children (MIS-C) but not during mild disease. In olah-/- mice, lethal influenza infection led to survival and mild disease as well as reduced lung viral loads, tissue damage, infection-driven pulmonary cell infiltration, and inflammation. This was underpinned by differential lipid droplet dynamics as well as reduced viral replication and virus-induced inflammation in macrophages. Supplementation of oleic acid, the main product of OLAH, increased influenza replication in macrophages and their inflammatory potential. Our findings define how the expression of OLAH drives life-threatening viral disease.

A three-stage intrathymic development pathway for the mucosal-associated invariant T cell lineage.

Mucosal-associated invariant T cells (MAIT cells) detect microbial vitamin B2 derivatives presented by the antigen-presenting molecule MR1. Here we defined three developmental stages and checkpoints for the MAIT cell lineage in humans and mice. Stage 1 and stage 2 MAIT cells predominated in thymus, while stage 3 cells progressively increased in abundance extrathymically. Transition through each checkpoint was regulated by MR1, whereas the final checkpoint that generated mature functional MAIT cells was controlled by multiple factors, including the transcription factor PLZF and microbial colonization. Furthermore, stage 3 MAIT cell populations were expanded in mice deficient in the antigen-presenting molecule CD1d, suggestive of a niche shared by MAIT cells and natural killer T cells (NKT cells). Accordingly, this study maps the developmental pathway and checkpoints that control the generation of functional MAIT cells.

Effect of Hydralazine on Angiotensin II-Induced Abdominal Aortic Aneurysm in Apolipoprotein E-Deficient Mice.

The rupture of an abdominal aortic aneurysm (AAA) causes about 200,000 deaths worldwide each year. However, there are currently no effective drug therapies to prevent AAA formation or, when present, to decrease progression and rupture, highlighting an urgent need for more research in this field. Increased vascular inflammation and enhanced apoptosis of vascular smooth muscle cells (VSMCs) are implicated in AAA formation. Here, we investigated whether hydralazine, which has anti-inflammatory and anti-apoptotic properties, inhibited AAA formation and pathological hallmarks. In cultured VSMCs, hydralazine (100 µM) inhibited the increase in inflammatory gene expression and apoptosis induced by acrolein and hydrogen peroxide, two oxidants that may play a role in AAA pathogenesis. The anti-apoptotic effect of hydralazine was associated with a decrease in caspase 8 gene expression. In a mouse model of AAA induced by subcutaneous angiotensin II infusion (1 µg/kg body weight/min) for 28 days in apolipoprotein E-deficient mice, hydralazine treatment (24 mg/kg/day) significantly decreased AAA incidence from 80% to 20% and suprarenal aortic diameter by 32% from 2.26 mm to 1.53 mm. Hydralazine treatment also significantly increased the survival rate from 60% to 100%. In conclusion, hydralazine inhibited AAA formation and rupture in a mouse model, which was associated with its anti-inflammatory and anti-apoptotic properties.

A high-dimensional cytometry atlas of peripheral blood over the human life span.

Age can profoundly affect susceptibility to a broad range of human diseases. Children are more susceptible to some infectious diseases such as diphtheria and pertussis, while in others, such as coronavirus disease 2019 and hepatitis A, they are more protected compared with adults. One explanation is that the composition of the immune system is a major contributing factor to disease susceptibility and severity. While most studies of the human immune system have focused on adults, how the immune system changes after birth remains poorly understood. Here, using high-dimensional spectral flow cytometry and computational methods for data integration, we analyzed more than 50 populations of immune cells in the peripheral blood, generating an immune cell atlas that defines the healthy human immune system from birth up to 75 years of age. We focused our efforts on children under 18 years old, revealing major changes in immune cell populations after birth and in children of schooling age. Specifically, CD4+ T effector memory cells, Vδ2+ gamma delta (γδ)T cells, memory B cells, plasmablasts, CD11c+ B cells and CD16+ CD56bright natural killer (NK) cells peaked in children aged 5-9 years old, whereas frequencies of T helper 1, T helper 17, dendritic cells and CD16+ CD57+ CD56dim NK cells were highest in older children (10-18 years old). The frequency of mucosal-associated invariant T cells was low in the first several years of life and highest in adults between 19 and 30 years old. Late adulthood was associated with fewer mucosal-associated invariant T cells and Vδ2+ γδ T cells but with increased frequencies of memory subsets of B cells, CD4+ and CD8+ T cells and CD57+ NK cells. This human immune cell atlas provides a critical resource to understand changes to the immune system during life and provides a reference for investigating the immune system in the context of human disease. This work may also help guide future therapies that target specific populations of immune cells to protect at-risk populations.

The attributes of plakins in cancer and disease: perspectives on ovarian cancer progression, chemoresistance and recurrence.

The plakin family of cytoskeletal proteins play an important role in cancer progression yet are under-studied in cancer, especially ovarian cancer. These large cytoskeletal proteins have primary roles in the maintenance of cytoskeletal integrity but are also associated with scaffolds of intermediate filaments and hemidesmosomal adhesion complexes mediating signalling pathways that regulate cellular growth, migration, invasion and differentiation as well as stress response. Abnormalities of plakins, and the closely related spectraplakins, result in diseases of the skin, striated muscle and nervous tissue. Their prevalence in epithelial cells suggests that plakins may play a role in epithelial ovarian cancer progression and recurrence. In this review article, we explore the roles of plakins, particularly plectin, periplakin and envoplakin in disease-states and cancers with emphasis on ovarian cancer. We discuss the potential role the plakin family of proteins play in regulating cancer cell growth, survival, migration, invasion and drug resistance. We highlight potential relationships between plakins, epithelial-mesenchymal transition (EMT) and cancer stem cells (CSCs) and discuss how interaction of these processes may affect ovarian cancer progression, chemoresistance and ultimately recurrence. We propose that molecular changes in the expression of plakins leads to the transition of benign ovarian tumours to carcinomas, as well as floating cellular aggregates (commonly known as spheroids) in the ascites microenvironment, which may contribute to the sustenance and progression of the disease. In this review, attempts have been made to understand the crucial changes in plakin expression in relation to progression and recurrence of ovarian cancer. Video Abstract.

The BH3-only proteins Bim and Puma cooperate to impose deletional tolerance of organ-specific antigens.

Although the proapoptotic BH3-only protein, Bim, is required for deletion of autoreactive thymocytes, Bim-deficient mice do not succumb to extensive organ-specific autoimmune disease. To determine whether other BH3-only proteins safeguard tolerance in the absence of Bim, we screened mice lacking Bim as well as other BH3-only proteins. Most strains showed no additional defects; however, mice deficient for both Puma and Bim spontaneously developed autoimmunity in multiple organs, and their T cells could transfer organ-specific autoimmunity. Puma- and Bim-double-deficient mice had a striking accumulation of mature, single-positive thymocytes, suggesting an additional defect in thymic deletion was the basis for disease. Transgenic mouse models of thymocyte deletion by peripheral neoantigens confirmed that the loss of Bim and Puma allowed increased numbers of autoreactive thymocytes to escape deletion. Our data show that Puma cooperates with Bim to impose a thymic-deletion checkpoint to peripheral self-antigens and cement the notion that defects in apoptosis alone are sufficient to cause autoimmune disease.

Foxp3+ Tregs from Langerhans cell histiocytosis lesions co-express CD56 and have a definitively regulatory capacity.

Langerhans cell histiocytosis (LCH) lesions contain myeloid lineage 'LCH' cells. Regulatory T cells (Tregs) are also enriched within lesions, although their role in LCH pathogenesis is unknown. LCH cells are thought to produce the transforming growth factor beta (TGF-ß) within lesions, however whether Tregs contribute is unestablished. Using flow cytometry, we analyzed relative frequencies of live Tregs from LCH patients and identified CD56 expression and TGF-ß production by lesion Tregs. While CD56+ Tregs were enriched in lesions, overall CD56+ T cells were reduced in the blood from active LCH patients compared to non-active disease patients, and there was a negative correlation between CD8+CD56+ T cells and Tregs. We propose that inducing a Treg phenotype in T cells such as CD56+ T cells may be a mechanism by which LCH cells divert inflammatory T cell responses. Thus, Tregs within LCH lesions are likely an important component in LCH pathogenesis.

Chronically stimulated human MAIT cells are unexpectedly potent IL-13 producers.

Mucosal-associated invariant T (MAIT) cells are unconventional T cells that recognize antigens derived from riboflavin biosynthesis. In addition to anti-microbial functions, human MAIT cells are associated with cancers, autoimmunity, allergies and inflammatory disorders, although their role is poorly understood. Activated MAIT cells are well known for their rapid release of Th1 and Th17 cytokines, but we have discovered that chronic stimulation can also lead to potent interleukin (IL)-13 expression. We used RNA-seq and qRT-PCR to demonstrate high expression of the IL-13 gene in chronically stimulated MAIT cells, and directly identify IL-13 using intracellular flow cytometry and multiplex bead analysis of MAIT cell cultures. This unexpected finding has important implications for IL-13-dependent diseases, such as colorectal cancer (CRC), that occur in mucosal areas where MAIT cells are abundant. We identify MAIT cells near CRC tumors and show that these areas and precancerous polyps express high levels of the IL-13 receptor, which promotes tumor progression and metastasis. Our data suggest that MAIT cells have a more complicated role in CRC than currently realized and that they represent a promising new target for immunotherapies where IL-13 can be a critical factor.

Divergent SATB1 expression across human life span and tissue compartments.

Special AT-rich binding protein-1 (SATB1) is a global chromatin organizer capable of activating or repressing gene transcription in mice and humans. The role of SATB1 is pivotal for T-cell development, with SATB1-knockout mice being neonatally lethal, although the exact mechanism is unknown. Moreover, SATB1 is dysregulated in T-cell lymphoma and proposed to suppress transcription of the Pdcd1 gene, encoding the immune checkpoint programmed cell death protein 1 (PD-1). Thus, SATB1 expression in T-cell subsets across different tissue compartments in humans is of potential importance for targeting PD-1. Here, we comprehensively analyzed SATB1 expression across different human tissues and immune compartments by flow cytometry and correlated this with PD-1 expression. We investigated SATB1 protein levels in pediatric and adult donors and assessed expression dynamics of this chromatin organizer across different immune cell subsets in human organs, as well as in antigen-specific T cells directed against acute and chronic viral infections. Our data demonstrate that SATB1 expression in humans is the highest in T-cell progenitors in the thymus, and then becomes downregulated in mature T cells in the periphery. Importantly, SATB1 expression in peripheral mature T cells is not static and follows fine-tuned expression dynamics, which appear to be tissue- and antigen-dependent. Furthermore, SATB1 expression negatively correlates with PD-1 expression in virus-specific CD8+ T cells. Our study has implications for understanding the role of SATB1 in human health and disease and suggests an approach for modulating PD-1 in T cells, highly relevant to human malignancies or chronic viral infections.

A potentially important role for T cells and regulatory T cells in Langerhans cell histiocytosis.

Langerhans cell histiocytosis is characterized by lesions containing inflammatory immune cells, including myeloid cells and T cells. Patient mortality remains unacceptably high and new treatment options are required. Several LCH studies have identified aberrant frequencies of T cell subsets with potential immune regulatory properties. High numbers of Foxp3+ regulatory T cells and gamma-delta T cells have been reported in patients with LCH, although, the cause of their presence or their significance is not yet clear. This review describes the current understanding of how LCH develops and progresses, focusing on the growing evidence that regulatory T cell subsets may be important and discussing the exciting potential for harnessing these cells to treat LCH using immune based therapies.

Human blood MAIT cell subsets defined using MR1 tetramers.

Mucosal-associated invariant T (MAIT) cells represent up to 10% of circulating human T cells. They are usually defined using combinations of non-lineage-specific (surrogate) markers such as anti-TRAV1-2, CD161, IL-18Rα and CD26. The development of MR1-Ag tetramers now permits the specific identification of MAIT cells based on T-cell receptor specificity. Here, we compare these approaches for identifying MAIT cells and show that surrogate markers are not always accurate in identifying these cells, particularly the CD4+ fraction. Moreover, while all MAIT cell subsets produced comparable levels of IFNγ, TNF and IL-17A, the CD4+ population produced more IL-2 than the other subsets. In a human ontogeny study, we show that the frequencies of most MR1 tetramer+ MAIT cells, with the exception of CD4+ MAIT cells, increased from birth to about 25 years of age and declined thereafter. We also demonstrate a positive association between the frequency of MAIT cells and other unconventional T cells including Natural Killer T (NKT) cells and Vδ2+ γδ T cells. Accordingly, this study demonstrates that MAIT cells are phenotypically and functionally diverse, that surrogate markers may not reliably identify all of these cells, and that their numbers are regulated in an age-dependent manner and correlate with NKT and Vδ2+ γδ T cells.

Control points in NKT-cell development.

CD1d-dependent natural killer T (NKT) cells are a unique T-cell subset with the ability to regulate the immune system in response to a broad range of diseases. That low NKT-cell numbers are associated with many different disease states in mice and humans, combined with the fact that NKT-cell numbers vary widely between individuals, makes it crucial to understand how these cells develop and how their numbers are maintained. Here, we review the current state of knowledge of NKT-cell development and attempt to highlight the most important questions in this field.

The NF-κB1 transcription factor prevents the intrathymic development of CD8 T cells with memory properties.

The role of specific members of the NF-κB family of transcription factors in CD8 T-cell selection and development is largely unknown. Here, we show that mice lacking NF-κB1 develop a unique population of conventional CD8 single-positive (SP) thymocytes with memory T cell-like properties that populate peripheral immune organs. Development of this memory-like population is not due to PLZF(+) thymocytes and instead coincides with changes in CD8 T-cell selection. These include a reduction in the efficiency of negative selection and a dependence on MHC class Ia or Ib expressed by haematopoietic cells. These findings indicate that NF-κB1 regulates multiple events in the thymus that collectively inhibit the excess development of CD8(+) thymocytes with memory cell characteristics.

The glucocorticoid receptor 1A3 promoter correlates with high sensitivity to glucocorticoid-induced apoptosis in human lymphocytes.

Glucocorticoids (GCs) are powerful inhibitors of inflammation and immunity. Although glucocorticoid-induced cell death (GICD) is an important part of GCs actions, the cell types and molecular mechanisms involved are not well understood. Untranslated exon 1A3 of the human glucocorticoid receptor (GR) gene is a major determinant of GICD in GICD-sensitive human cancer cell lines, operating to dynamically upregulate GR levels in response to GCs. We measured the GICD sensitivity of freshly isolated peripheral blood mononuclear cells and thymocytes to dexamethasone in vitro, relating this to GR exon 1A3 expression. A clear GICD sensitivity hierarchy was detected: B cells>thymocytes/natural killer (NK) cells>peripheral T cells. Within thymocyte populations, GICD sensitivity decreased with maturation. Interestingly, NK cell subsets were differentially sensitive to GICD, with CD16(+)CD56(int) (cytotoxic) NK cells being highly resistant to GICD, whereas CD16(-)CD56(hi) (cytokine producing) NK cells were highly sensitive (similar to B cells). B-cell GICD was rescued by co-culture with interleukin-4. Strikingly, although no significant increases in GR protein were observed during 48 h of culture of GICD-sensitive and -resistant cells alike, GR 1A3 expression was increased over pre-culture levels in a manner directly proportional to the GICD sensitivity of each cell type. Accordingly, this is the first evidence that the GR exon 1A3 promoter is differentially regulated during thymic development and maturation of human T cells. Furthermore, human peripheral blood B cells are exquisitely GICD-sensitive in vitro, giving new insight into how GCs may downregulate immunity. Collectively, these data show that GR 1A3 expression is tied with GICD sensitivity in human lymphocytes, underscoring the potential for GR 1A3 expression to be used as a biomarker for sensitivity to GICD.

Thymic development of human natural killer T cells: recent advances and implications for immunotherapy.

Invariant natural killer T (iNKT) cells are a subset of lipid-reactive, unconventional T cells that have anti-tumor properties that make them a promising target for cancer immunotherapy. Recent studies have deciphered the developmental pathway of human MAIT and Vγ9Vδ2 γδ-T cells as well as murine iNKT cells, yet our understanding of human NKT cell development is limited. Here, we provide an update in our understanding of how NKT cells develop in the human body and how knowledge regarding their development could enhance human treatments by targeting these cells.

Methodological optimisation of thymocyte isolation and cryopreservation of human thymus samples.

Premature lymphocytes develop into non-autoreactive, mature naïve CD4+ or CD8+ T cells in the thymus before entering the circulation. However, in-depth characterization of human thymocyte development remains challenging due to limited availability of human thymus samples and the fragile nature of thymocyte populations. Thymocytes often do not survive cryopreservation and thawing procedures, especially the fragile CD4+CD8+ double positive population. It is generally recommended to use fresh human thymus tissue on the day of excision to avoid any biases in thymocyte composition. This hampers the possibility to perform multiple experiments on the same thymus sample. To establish how the thymocyte viability and composition can be maintained, we compared two thymocyte isolation methods used for human and/or mice thymi, three cryopreservation methods in combination with our most gentle thawing technique. Based on our findings we established that fresh human thymi remain viable in cold storage for up to two days post-surgery without compromising thymocyte composition. Thymocytes can be cryopreserved if required, although the CD4+CD8+ double positive populations may be reduced. Our study provides thoroughly optimized methods to study human thymocyte development over a considerable time-frame post-surgery.

Distinct subpopulations of DN1 thymocytes exhibit preferential γδ T lineage potential.

The αß and γδ T cell lineages both differentiate in the thymus from common uncommitted progenitors. The earliest stage of T cell development is known as CD4-CD8- double negative 1 (DN1), which has previously been shown to be a heterogenous mixture of cells. Of these, only the CD117+ fraction has been proposed to be true T cell progenitors that progress to the DN2 and DN3 thymocyte stages, at which point the development of the αß and γδ T cell lineages diverge. However, recently, it has been shown that at least some γδ T cells may be derived from a subset of CD117- DN thymocytes. Along with other ambiguities, this suggests that T cell development may not be as straightforward as previously thought. To better understand early T cell development, particularly the heterogeneity of DN1 thymocytes, we performed a single cell RNA sequence (scRNAseq) of mouse DN and γδ thymocytes and show that the various DN stages indeed comprise a transcriptionally diverse subpopulations of cells. We also show that multiple subpopulations of DN1 thymocytes exhibit preferential development towards the γδ lineage. Furthermore, specific γδ-primed DN1 subpopulations preferentially develop into IL-17 or IFNγ-producing γδ T cells. We show that DN1 subpopulations that only give rise to IL-17-producing γδ T cells already express many of the transcription factors associated with type 17 immune cell responses, while the DN1 subpopulations that can give rise to IFNγ-producing γδ T cell already express transcription factors associated with type 1 immune cell responses.

A three-stage developmental pathway for human Vγ9Vδ2 T cells within the postnatal thymus.

Vγ9Vδ2 T cells are the largest population of γδ T cells in adults and can play important roles in providing effective immunity against cancer and infection. Many studies have suggested that peripheral Vγ9Vδ2 T cells are derived from the fetal liver and thymus and that the postnatal thymus plays little role in the development of these cells. More recent evidence suggested that these cells may also develop postnatally in the thymus. Here, we used high-dimensional flow cytometry, transcriptomic analysis, functional assays, and precursor-product experiments to define the development pathway of Vγ9Vδ2 T cells in the postnatal thymus. We identify three distinct stages of development for Vγ9Vδ2 T cells in the postnatal thymus that are defined by the progressive acquisition of functional potential and major changes in the expression of transcription factors, chemokines, and other surface markers. Furthermore, our analysis of donor-matched thymus and blood revealed that the molecular requirements for the development of functional Vγ9Vδ2 T cells are delivered predominantly by the postnatal thymus and not in the periphery. Tbet and Eomes, which are required for IFN-γ and TNFα expression, are up-regulated as Vγ9Vδ2 T cells mature in the thymus, and mature thymic Vγ9Vδ2 T cells rapidly express high levels of these cytokines after stimulation. Similarly, the postnatal thymus programs Vγ9Vδ2 T cells to express the cytolytic molecules, perforin, granzyme A, and granzyme K. This study provides a greater understanding of how Vγ9Vδ2 T cells develop in humans and may lead to opportunities to manipulate these cells to treat human diseases.

Divergent molecular networks program functionally distinct CD8+ skin-resident memory T cells.

Skin-resident CD8+ T cells include distinct interferon-γ-producing [tissue-resident memory T type 1 (TRM1)] and interleukin-17 (IL-17)-producing (TRM17) subsets that differentially contribute to immune responses. However, whether these populations use common mechanisms to establish tissue residence is unknown. In this work, we show that TRM1 and TRM17 cells navigate divergent trajectories to acquire tissue residency in the skin. TRM1 cells depend on a T-bet-Hobit-IL-15 axis, whereas TRM17 cells develop independently of these factors. Instead, c-Maf commands a tissue-resident program in TRM17 cells parallel to that induced by Hobit in TRM1 cells, with an ICOS-c-Maf-IL-7 axis pivotal to TRM17 cell commitment. Accordingly, by targeting this pathway, skin TRM17 cells can be ablated without compromising their TRM1 counterparts. Thus, skin-resident T cells rely on distinct molecular circuitries, which can be exploited to strategically modulate local immunity.