Reactive oxygen species (ROS) are critical in driving the onset of type 1 diabetes (T1D). Ablation of ROS derived from phagocytic NADPH oxidase 2 is protective against autoimmune diabetes in ...non-obese diabetic (NOD
mice. However, the mechanisms of NADPH oxidase 2-derived ROS in T1D pathogenesis need to be elucidated. Here, we have examined the role of
(the regulatory subunit of NADPH oxidase 2) in dendritic cells (DC).
-mutant DCs exhibit reduced ability to activate autoreactive CD8
T cells despite no difference in co-stimulatory molecule expression or pro-inflammatory cytokine production. When provided with exogenous whole-protein antigen,
-mutant NOD DCs showed strong phagosome acidification and rapid antigen degradation, which lead to an absence of protein translocation into the cytoplasm and deficient antigenic peptide loading on MHC Class I molecules.
This study demonstrates that Ncf1 (p47
) is required for activation and effector function of CD8
T cells by acting both intrinsically within the T cell as well as within professional antigen presenting cells.
ROS promote CD8
T cell activation by facilitating autoantigen cross-presentation by DCs. ROS scavengers could potentially represent an important component of therapies aiming to disrupt autoantigen presentation and activation of CD8
T cells in individuals at-risk for developing T1D.
Sepsis engenders distinct host immunologic changes that include the expansion of myeloid-derived suppressor cells (MDSCs). These cells play a physiologic role in tempering acute inflammatory ...responses but can persist in patients who develop chronic critical illness.
Cellular Indexing of Transcriptomes and Epitopes by Sequencing and transcriptomic analysis are used to describe MDSC subpopulations based on differential gene expression, RNA velocities, and biologic process clustering.
We identify a unique lineage and differentiation pathway for MDSCs after sepsis and describe a novel MDSC subpopulation. Additionally, we report that the heterogeneous response of the myeloid compartment of blood to sepsis is dependent on clinical outcome.
The origins and lineage of these MDSC subpopulations were previously assumed to be discrete and unidirectional; however, these cells exhibit a dynamic phenotype with considerable plasticity.
Low-dose antithymocyte globulin (ATG) plus pegylated granulocyte colony-stimulating factor (G-CSF) preserves β-cell function for at least 12 months in type 1 diabetes. Herein, we describe metabolic ...and immunological parameters 24 months following treatment. Patients with established type 1 diabetes (duration 4-24 months) were randomized to ATG and pegylated G-CSF (ATG+G-CSF) (N = 17) or placebo (N = 8). Primary outcomes included C-peptide area under the curve (AUC) following a mixed-meal tolerance test (MMTT) and flow cytometry. "Responders" (12-month C-peptide ≥ baseline), "super responders" (24-month C-peptide ≥ baseline), and "nonresponders" (12-month C-peptide < baseline) were evaluated for biomarkers of outcome. At 24 months, MMTT-stimulated AUC C-peptide was not significantly different in ATG+G-CSF (0.49 nmol/L/min) versus placebo (0.29 nmol/L/min). Subjects treated with ATG+G-CSF demonstrated reduced CD4
T cells and CD4
/CD8
T-cell ratio and increased CD16
CD56
natural killer cells (NK), CD4
effector memory T cells (Tem), CD4
PD-1
central memory T cells (Tcm), Tcm PD-1 expression, and neutrophils. FOXP3
Helios
regulatory T cells (Treg) were elevated in ATG+G-CSF subjects at 6, 12, and 18 but not 24 months. Immunophenotyping identified differential HLA-DR expression on monocytes and NK and altered CXCR3 and PD-1 expression on T-cell subsets. As such, a group of metabolic and immunological responders was identified. A phase II study of ATG+G-CSF in patients with new-onset type 1 diabetes is ongoing and may support ATG+G-CSF as a prevention strategy in high-risk subjects.
Previously, we demonstrated low-dose antithymocyte globulin (ATG) and granulocyte colony-stimulating factor (GCSF) immunotherapy preserved C-peptide for 2 years in a pilot study of patients with ...established type 1 diabetes (
= 25). Here, we evaluated the long-term outcomes of ATG/GCSF in study participants with 5 years of available follow-up data (
= 15). The primary end point was area under the curve (AUC) C-peptide during a 2-h mixed-meal tolerance test. After 5 years, there were no statistically significant differences in AUC C-peptide when comparing those who received ATG/GCSF versus placebo (
= 0.41). A modeling framework based on mean trajectories in C-peptide AUC over 5 years, accounting for differing trends between groups, was applied to recategorize responders (
= 9) and nonresponders (
= 7). ATG/GCSF reponders demonstrated nearly unchanged HbA
over 5 years (mean 95% CI adjusted change 0.29% -0.69%, 1.27%), but the study was not powered for comparisons against nonresponders 1.75% (-0.57%, 4.06%) or placebo recipients 1.44% (0.21%, 2.66%). These data underscore the importance of long-term follow-up in previous and ongoing phase 2 trials of low-dose ATG in recent-onset type 1 diabetes.
IL-12 and IL-18 synergize to promote TH1 responses and have been implicated as accelerators of autoimmune pathogenesis in type 1 diabetes (T1D). We investigated the influence of these cytokines on ...immune cells involved in human T1D progression: natural killer (NK) cells, regulatory T cells (Tregs), and cytotoxic T lymphocytes (CTL). NK cells from T1D patients exhibited higher surface CD226 versus controls and lower CD25 compared to first-degree relatives and controls. Changes in NK cell phenotype towards terminal differentiation were associated with cytomegalovirus (CMV) seropositivity, while possession of IL18RAP, IFIH1, and IL2RA T1D-risk variants impacted NK cell activation as evaluated by immuno-expression quantitative trait loci (eQTL) analyses. IL-12 and IL-18 stimulated NK cells from healthy donors exhibited enhanced specific killing of myelogenous K562 target cells. Moreover, activated NK cells increased expression of NKG2A, NKG2D, CD226, TIGIT and CD25, which enabled competition for IL-2 upon co-culture with Tregs, resulting in Treg downregulation of FOXP3, production of IFNγ, and loss of suppressive function. We generated islet-autoreactive CTL “avatars”, which upon exposure to IL-12 and IL-18, upregulated IFNγ and Granzyme-B leading to increased lymphocytotoxicity of a human β-cell line in vitro. These results support a model for T1D pathogenesis wherein IL-12 and IL-18 synergistically enhance CTL and NK cell cytotoxic activity and disrupt immunoregulation by Tregs.
Working Model Summarizing the Hypothesized Contributions of Elevated IL-12 and IL-18 Levels Toward Failure in Immunoregulation and T1D Pathogenesis. In immune homeostasis (left), regulatory T cells (Treg) suppress activation and function of CD8+T cells, CD4+T cells and NK cells via various mechanisms including competition for IL-2. In settings of increased genetic risk for T1D, exposure to some environmental trigger(s) compound genetic defects to induce a break in tolerance (right), during which time IL-12 and IL-18 levels are elevated and NK cells upregulate CD25. This allows for direct competition with Tregs for IL-2, resulting in decreased Treg IL-2 signaling, STAT5 phosphorylation (pSTAT5), and FOXP3 expression, ultimately abrogating suppression. We hypothesize that this, together with enhanced production of cytokines and cytolytic proteins by CD4+conventional T cells and CD8+cytotoxic T cells, leads to augmented lysis of β-cells (right). Display omitted
•IL-12 & IL-18 enhance NK cell and antigen specific CD8 T cell killing.•Regulatory T cells (Tregs) lose suppressive capacity and produce IFN-γ.•Type 1 Diabetes candidate genes permit CD25 upregulation on NK cells.•NK cells exhibit altered receptor balance and are able to compete with Tregs for IL-2 in vitro.
A missense mutation (R620W) of protein tyrosine phosphatase nonreceptor type 22 (
), which encodes lymphoid-tyrosine phosphatase (LYP), confers genetic risk for multiple autoimmune diseases including ...type 1 diabetes. LYP has been putatively demonstrated to attenuate proximal T and BCR signaling. However, limited data exist regarding
expression within primary T cell subsets and the impact of the type 1 diabetes risk variant on human T cell activity. In this study, we demonstrate endogenous
is differentially expressed and dynamically controlled following activation. From control subjects homozygous for the nonrisk allele, we observed 2.1- (
< 0.05) and 3.6-fold (
< 0.001) more
transcripts in resting CD4
memory and regulatory T cells (Tregs), respectively, over naive CD4
T cells, with expression peaking 24 h postactivation. When LYP was overexpressed in conventional CD4
T cells, TCR signaling and activation were blunted by LYP-620R (
< 0.001) but only modestly affected by the LYP-620W risk variant versus mock-transfected control, with similar results observed in Tregs. LYP overexpression only impacted proliferation following activation by APCs but not anti-CD3- and anti-CD28-coated microbeads, suggesting LYP modulation of pathways other than TCR. Notably, proliferation was significantly lower with LYP-620R than with LYP-620W overexpression in conventional CD4
T cells but was similar in Treg. These data indicate that the LYP-620W variant is hypomorphic in the context of human CD4
T cell activation and may have important implications for therapies seeking to restore immunological tolerance in autoimmune disorders.
NKG2D is implicated in autoimmune diabetes. However, the role of this receptor in diabetes pathogenesis is unclear owing to conflicting results with studies involving global inhibition of NKG2D ...signaling. We found that NKG2D and its ligands are present in human pancreata, with expression of NKG2D and its ligands increased in the islets of patients with type 1 diabetes. To directly assess the role of NKG2D in the pancreas, we generated NOD mice that express an NKG2D ligand in β-islet cells. Diabetes was reduced in these mice. The reduction corresponded with a decrease in the effector to central memory CD8
T-cell ratio. Further, NKG2D signaling during in vitro activation of both mouse and human CD8
T cells resulted in an increased number of central memory CD8
T cells and diabetes protection by central memory CD8
T cells in vivo. Taken together, these studies demonstrate that there is a protective role for central memory CD8
T cells in autoimmune diabetes and that this protection is enhanced with NKG2D signaling. These findings stress the importance of anatomical location when determining the role NKG2D signaling plays, as well as when developing therapeutic strategies targeting this pathway, in type 1 diabetes development.
Innate immunity is directly associated with Type 1 diabetes (T1D) . Recently, N6-methyladenosine (m6A) has been shown to modulate human β-cell biology and innate immunity. Here, we focused on the ...contribution of m6A in modulating T1D development. RNA-seq. in β-cells revealed downregulation of the m6A writers (Mettl3, Mettl14, and Wtap) in pre-diabetic NOD mice. Consistently, pancreases from female NOD mice and human T1D showed downregulation of METTL3 protein levels in insulin-positive cells as the disease progressed. m6A-sequencing in human T1D versus control islets and intersection with differentially expressed genes (DEGs) in T1D β-cells from a single-cell RNA seq. dataset, revealed enriched pathways associated with ER function, T1D, and apoptosis. Pro-inflammatory cytokines are directly linked to β-cell autoimmunity and are considered a useful tool to study T1D onset. We applied m6A-sequencing in cytokine-treated human islet preparations, and in parallel studies, treated EndoC-βH1 cells with cytokines followed by RNA-sequencing. Intersection of m6A-decorated and common DEGs in human islets and EndoC-βH1 cells revealed enrichment in pathways associated with the innate immune response. METTL3 silencing in EndoC-βH1 impacted several innate immune mediators upon cytokine challenge. Incubation of human islets from multiple donors with ER-stress inducers, ROS donors, NO donors, and ROS or NO scavengers revealed that pre-existing ER stress and ROS decrease METTL3, while physiological levels of NO upregulate METTL3. We conclude that ROS drives METTL3 downregulation associated with T1D. Exploring therapeutic relevance by AAV-8 mediated gene delivery showed that β-cell specific upregulation of Mettl3 leads to a decrease in hyperglycemia during early stages of T1D in female NOD mice. Together, these results demonstrate a novel layer of regulation in human β-cells in the context of T1D and point to targeting METTL3 as a therapeutic approach to promote β-cell survival and improve glycemia.
Disclosure
D.F.De jesus: n/a. D.L.Eizirik: None. R.Kulkarni: Advisory Panel; Biomea, Novo Nordisk, REDD Pharma, Consultant; Relay Therapeutics, Research Support; Inversago, SerPlus. Z.Zhang: Research Support; SinoVac. N.K.Brown: None. G.Basile: None. L.Xiao: None. S.Kahraman: Employee; Boehringer Ingelheim International GmbH. C.E.Mathews: None. A.C.Powers: None. M.A.Atkinson: None.
Funding
American Diabetes Association (7-21-PDF-140) ; R01 DK067536R01 DK103215R01 DK117639
Multidimensional peptide separations can greatly increase the depth of coverage in proteome profiling. However, a major challenge for multidimensional separations is the requirement of large ...biological samples, often containing milligram amounts of protein. We have developed nanowell-mediated two-dimensional (2D) reversed-phase nanoflow liquid chromatography (LC) separations for in-depth proteome profiling of low-nanogram samples. Peptides are first separated using high-pH LC and the effluent is concatenated into 4 or 12 nanowells. The contents of each nanowell are reconstituted in LC buffer and collected for subsequent separation and analysis by low-pH nanoLC-MS/MS. The nanowell platform minimizes peptide losses to surfaces in offline 2D LC fractionation, enabling >5800 proteins to be confidently identified from just 50 ng of HeLa digest. Furthermore, in combination with a recently developed nanowell-based sample preparation workflow, we demonstrated deep proteome profiling of >6000 protein groups from small populations of cells, including ∼650 HeLa cells and 10 single human pancreatic islet thin sections (∼1000 cells) from a pre-symptomatic type 1 diabetic donor.
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