Feasibility and Validity of In-Home Self-Collected Capillary Blood Spot Screening for Type 1 Diabetes Risk.

Aims: Self-collection of a blood sample for autoantibody testing has potential to facilitate screening for type 1 diabetes risk. We sought to determine the feasibility and acceptability of this approach and the performance of downstream antibody assays. Methods: People living with type 1 diabetes and their family members (N = 97) provided paired capillary blood spot and serum samples collected, respectively, by themselves and a health worker. They provided feedback on the ease, convenience, and painfulness of blood spot collection. Islet antibodies were measured in blood spots by antibody detection by agglutination PCR (ADAP) or multiplex enzyme-linked immunoassay (ELISA), and in serum by radioimmunoassay (RIA) or ELISA. Results: Using serum RIA and ELISA to define antibody status, 50 antibody-negative (Abneg) and 47 antibody-positive (Abpos) participants enrolled, of whom 43 and 47, respectively, returned testable blood spot samples. The majority indicated that self-collection was easier, more convenient, and less painful than formal venesection. The sensitivity and specificity for detection of Abpos by blood spot were, respectively, 85% and 98% for ADAP and 87% and 100% for multiplex ELISA. The specificities by ADAP for each of the four antigen specificities ranged from 98% to 100% and areas under the receiver operator curve from 0.841 to 0.986. Conclusions: Self-collected blood spot sampling is preferred over venesection by research participants. ADAP and multiplex ELISA are highly specific assays for islet antibodies in blood spots with acceptable performance for use alone or in combination to facilitate screening for type 1 diabetes risk. Clinical Trial Registration number: ACTRN12620000510943.

Human HLA-DR+CD27+ regulatory T cells show enhanced antigen-specific suppressive function.

Regulatory T cells (Tregs) have potential for the treatment of autoimmune diseases and graft rejection. Antigen specificity and functional stability are considered critical for their therapeutic efficacy. In this study, expansion of human Tregs in the presence of porcine PBMCs (xenoantigen-expanded Tregs, Xn-Treg) allowed the selection of a distinct Treg subset, coexpressing the activation/memory surface markers HLA-DR and CD27 with enhanced proportion of FOXP3+Helios+ Tregs. Compared with their unsorted and HLA-DR+CD27+ double-positive (DP) cell-depleted Xn-Treg counterparts, HLA-DR+CD27+ DP-enriched Xn-Tregs expressed upregulated Treg function markers CD95 and ICOS with enhanced suppression of xenogeneic but not polyclonal mixed lymphocyte reaction. They also had less Treg-specific demethylation in the region of FOXP3 and were more resistant to conversion to effector cells under inflammatory conditions. Adoptive transfer of porcine islet recipient NOD/SCID IL2 receptor γ-/- mice with HLA-DR+CD27+ DP-enriched Xn-Tregs in a humanized mouse model inhibited porcine islet graft rejection mediated by 25-fold more human effector cells. The prolonged graft survival was associated with enhanced accumulation of FOXP3+ Tregs and upregulated expression of Treg functional genes, IL10 and cytotoxic T lymphocyte antigen 4, but downregulated expression of effector Th1, Th2, and Th17 cytokine genes, within surviving grafts. Collectively, human HLA-DR+CD27+ DP-enriched Xn-Tregs expressed a specific regulatory signature that enabled identification and isolation of antigen-specific and functionally stable Tregs with potential as a Treg-based therapy.

Cytotoxicity-Related Gene Expression and Chromatin Accessibility Define a Subset of CD4+ T Cells That Mark Progression to Type 1 Diabetes.

Type 1 diabetes in children is heralded by a preclinical phase defined by circulating autoantibodies to pancreatic islet antigens. How islet autoimmunity is initiated and then progresses to clinical diabetes remains poorly understood. Only one study has reported gene expression in specific immune cells of children at risk associated with progression to islet autoimmunity. We analyzed gene expression with RNA sequencing in CD4+ and CD8+ T cells, natural killer (NK) cells, and B cells, and chromatin accessibility by assay for transposase-accessible chromatin sequencing (ATAC-seq) in CD4+ T cells, in five genetically at risk children with islet autoantibodies who progressed to diabetes over a median of 3 years ("progressors") compared with five children matched for sex, age, and HLA-DR who had not progressed ("nonprogressors"). In progressors, differentially expressed genes (DEGs) were largely confined to CD4+ T cells and enriched for cytotoxicity-related genes/pathways. Several top-ranked DEGs were validated in a semi-independent cohort of 13 progressors and 11 nonprogressors. Flow cytometry confirmed that progression was associated with expansion of CD4+ cells with a cytotoxic phenotype. By ATAC-seq, progression was associated with reconfiguration of regulatory chromatin regions in CD4+ cells, some linked to differentially expressed cytotoxicity-related genes. Our findings suggest that cytotoxic CD4+ T cells play a role in promoting progression to type 1 diabetes.

Women with type 1 diabetes exhibit a progressive increase in gut Saccharomyces cerevisiae in pregnancy associated with evidence of gut inflammation.

AIMS: Studies of the gut microbiome have focused on its bacterial composition. We aimed to characterize the gut fungal microbiome (mycobiome) across pregnancy in women with and without type 1 diabetes. METHODS: Faecal samples (n = 162) were collected from 70 pregnant women (45 with and 25 without type 1 diabetes) across all trimesters. Fungi were analysed by internal transcribed spacer 1 amplicon sequencing. Markers of intestinal inflammation (faecal calprotectin) and intestinal epithelial integrity (serum intestinal fatty acid binding protein; I-FABP), and serum antibodies to Saccharomyces cerevisiae (ASCA) were measured. RESULTS: Women with type 1 diabetes had decreased fungal alpha diversity by the third trimester, associated with an increased abundance of Saccharomyces cerevisiae that was inversely related to the abundance of the anti-inflammatory butyrate-producing bacterium Faecalibacterium prausnitzii. Women with type 1 diabetes had higher concentrations of calprotectin, I-FABP and ASCA. CONCLUSIONS: Women with type 1 diabetes exhibit a shift in the gut mycobiome across pregnancy associated with evidence of gut inflammation and impaired intestinal barrier function. The relevance of these findings to the higher rate of pregnancy complications in type 1 diabetes warrants further study.

Chromosomes distribute randomly to, but not within, human neutrophil nuclear lobes.

The proximity pattern and radial distribution of chromosome territories within spherical nuclei are random and non-random, respectively. Whether this distribution pattern is conserved in the partitioned or lobed nuclei of polymorphonuclear cells is unclear. Here we use chromosome paint technology to examine the chromosome territories of all 46 chromosomes in hundreds of single human neutrophils - an abundant and famously polymorphonuclear immune cell. By comparing the distribution of chromosomes to randomly shuffled controls and validating with orthogonal chromosome conformation capture technology, we show for the first time that human chromosomes randomly distribute to neutrophil nuclear lobes, while maintaining a non-random radial distribution within these lobes. Furthermore, we demonstrate that chromosome length correlates with three-dimensional volume not only in neutrophils but other human immune cells. This work demonstrates that chromosomes are largely passive passengers during the neutrophil lobing process but are able to subsequently maintain their macro-level organization within lobes.

Multi-level remodelling of chromatin underlying activation of human T cells.

Remodelling of chromatin architecture is known to regulate gene expression and has been well characterized in cell lineage development but less so in response to cell perturbation. Activation of T cells, which triggers extensive changes in transcriptional programs, serves as an instructive model to elucidate how changes in chromatin architecture orchestrate gene expression in response to cell perturbation. To characterize coordinate changes at different levels of chromatin architecture, we analyzed chromatin accessibility, chromosome conformation and gene expression in activated human T cells. T cell activation was characterized by widespread changes in chromatin accessibility and interactions that were shared between activated CD4+ and CD8+ T cells, and with the formation of active regulatory regions associated with transcription factors relevant to T cell biology. Chromatin interactions that increased and decreased were coupled, respectively, with up- and down-regulation of corresponding target genes. Furthermore, activation was associated with disruption of long-range chromatin interactions and with partitioning of topologically associating domains (TADs) and remodelling of their TAD boundaries. Newly formed/strengthened TAD boundaries were associated with higher nucleosome occupancy and lower accessibility, linking changes in lower and higher order chromatin architecture. T cell activation exemplifies coordinate multi-level remodelling of chromatin underlying gene transcription.

Extreme disruption of heterochromatin is required for accelerated hematopoietic aging.

Loss of heterochromatin has been proposed as a universal mechanism of aging across different species and cell types. However, a comprehensive analysis of hematopoietic changes caused by heterochromatin loss is lacking. Moreover, there is conflict in the literature around the role of the major heterochromatic histone methyltransferase Suv39h1 in the aging process. Here, we use individual and dual deletion of Suv39h1 and Suv39h2 enzymes to examine the causal role of heterochromatin loss in hematopoietic cell development. Loss of neither Suv39h1 nor Suv39h2 individually had any effect on hematopoietic stem cell function or the development of mature lymphoid or myeloid lineages. However, deletion of both enzymes resulted in characteristic changes associated with aging such as reduced hematopoietic stem cell function, thymic involution and decreased lymphoid output with a skewing toward myeloid development, and increased memory T cells at the expense of naive T cells. These cellular changes were accompanied by molecular changes consistent with aging, including alterations in nuclear shape and increased nucleolar size. Together, our results indicate that the hematopoietic system has a remarkable tolerance for major disruptions in chromatin structure and reveal a role for Suv39h2 in depositing sufficient H3K9me3 to protect the entire hematopoietic system from changes associated with premature aging.

CD52 glycan binds the proinflammatory B box of HMGB1 to engage the Siglec-10 receptor and suppress human T cell function.

CD52, a glycophosphatidylinositol (GPI)-anchored glycoprotein, is released in a soluble form following T cell activation and binds to the Siglec (sialic acid-binding Ig-like lectin)-10 receptor on T cells to suppress their function. We show that binding of CD52-Fc to Siglec-10 and T cell suppression requires the damage-associated molecular pattern (DAMP) protein, high-mobility group box 1 (HMGB1). CD52-Fc bound specifically to the proinflammatory Box B domain of HMGB1, and this in turn promoted binding of the CD52 N-linked glycan, in α-2,3 sialic acid linkage with galactose, to Siglec-10. Suppression of T cell function was blocked by anti-HMGB1 antibody or the antiinflammatory Box A domain of HMGB1. CD52-Fc induced tyrosine phosphorylation of Siglec-10 and was recovered from T cells complexed with HMGB1 and Siglec-10 in association with SHP1 phosphatase and the T cell receptor (TCR). Thus, soluble CD52 exerts a concerted immunosuppressive effect by first sequestering HMGB1 to nullify its proinflammatory Box B, followed by binding to the inhibitory Siglec-10 receptor, triggering recruitment of SHP1 to the intracellular immunoreceptor tyrosine-based inhibitory motif of Siglec-10 and its interaction with the TCR. This mechanism may contribute to immune-inflammatory homeostasis in pathophysiologic states and underscores the potential of soluble CD52 as a therapeutic agent.

Cord Blood CD8+ T Cells Have a Natural Propensity to Express IL-4 in a Fatty Acid Metabolism and Caspase Activation-Dependent Manner.

How T cells differentiate in the neonate may critically determine the ability of the infant to cope with infections, respond to vaccines and avert allergies. Previously, we found that naïve cord blood CD4+ T cells differentiated toward an IL-4-expressing phenotype when activated in the presence of TGF-ß and monocyte-derived inflammatory cytokines, the latter are more highly secreted by infants who developed food allergy. Here, we show that in the absence of IL-2 or IL-12, naïve cord blood CD8+ T cells have a natural propensity to differentiate into IL-4-producing non-classic TC2 cells when they are activated alone, or in the presence of TGF-ß and/or inflammatory cytokines. Mechanistically, non-classic TC2 development is associated with decreased expression of IL-2 receptor alpha (CD25) and glycolysis, and increased fatty acid metabolism and caspase-dependent cell death. Consequently, the short chain fatty acid, sodium propionate (NaPo), enhanced IL-4 expression, but exogenous IL-2 or pan-caspase inhibition prevented IL-4 expression. In children with endoscopically and histologically confirmed non-inflammatory bowel disease and non-infectious pediatric idiopathic colitis, the presence of TGF-ß, NaPo, and IL-1ß or TNF-α promoted TC2 differentiation in vitro. In vivo, colonic mucosa of children with colitis had significantly increased expression of IL-4 in CD8+ T cells compared with controls. In addition, activated caspase-3 and IL-4 were co-expressed in CD8+ T cells in the colonic mucosa of children with colitis. Thus, in the context of colonic inflammation and limited IL-2 signaling, CD8+ T cells differentiate into non-classic TC2 that may contribute to the pathology of inflammatory/allergic diseases in children.

Genome-wide analysis reveals no evidence of trans chromosomal regulation of mammalian immune development.

It has been proposed that interactions between mammalian chromosomes, or transchromosomal interactions (also known as kissing chromosomes), regulate gene expression and cell fate determination. Here we aimed to identify novel transchromosomal interactions in immune cells by high-resolution genome-wide chromosome conformation capture. Although we readily identified stable interactions in cis, and also between centromeres and telomeres on different chromosomes, surprisingly we identified no gene regulatory transchromosomal interactions in either mouse or human cells, including previously described interactions. We suggest that advances in the chromosome conformation capture technique and the unbiased nature of this approach allow more reliable capture of interactions between chromosomes than previous methods. Overall our findings suggest that stable transchromosomal interactions that regulate gene expression are not present in mammalian immune cells and that lineage identity is governed by cis, not trans chromosomal interactions.