Features of type 1 diabetes (T1D) include loss of first-phase insulin secretion in response to glucose, declining c-peptide, and glucose intolerance; indicating β cell dysfunction. Here, we ...investigate the mechanisms of this dysfunction with the hypothesis that immune dysregulation during the early stages of T1D development impacts β cell dysfunction in pathways affecting glucose metabolism. The live pancreas tissue slice (LPTS) model is ideal for the study of T1D pathogenesis due to its preservation of the pancreatic environment. LPTS were generated from organ donors without diabetes or autoantibodies (ND, n=9) , donors positive for one or more autoantibodies without a diagnosis of T1D (AAb+, n=6) , and donors with T1D (T1D, n=4) . Imaging studies were conducted to assess the impact of T cell infiltration on β cell function through Ca2+ imaging and slice perifusion. Islets from ND and AAb+ donors had no insulitis and exhibited Ca2+ responses to both high glucose (HG) and potassium chloride (KCl) . A majority of β cell+ islets in tissue from donors with T1D had infiltrating T cells with several having insulitis. Islet HG response from donors with T1D was heterogenous (∼50% failed to respond) while all T1D islets exhibited responses to KCl. Insulin secretion was significantly higher in LPTS from ND donors than in donors with T1D (p=0.0079) . To determine the basis for this loss of function, we assessed transcriptomics of islets within these cohorts. When comparing gene expression levels in islets from ND versus T1D, significant differences were observed in genes involved in glycolysis (GAPDH, p=3.27x10-21) , the citric acid cycle components (OGDH, p=1.88x10-18) , and the electric transport chain (F1FoATP synthase, p=2.69x10-13) . The decreased expression of glucose metabolism genes during T1D development along with the loss of β cell function in LPTS demonstrates β cell dysfunction before their demise and may be a contributory mechanism towards the pathogenesis of disease.
Disclosure
M.Huber: None. M.Slak rupnik: None. D.M.Drotar: None. H.Hiller: None. M.Beery: None. I.Kusmartseva: None. M.A.Atkinson: None. E.Phelps: Research Support; Immunocore, Ltd. C.E.Mathews: None.
Funding
National Institutes of Health (5T32DK108736-03) National Institutes of Health (PO1 AI42288)
The original version of this article unfortunately contained a mistake in the author group section. Shuyao Zhang’s family name was misspelled as “Zheng”.
In both humans and NOD mice, type 1 diabetes (T1D) develops from the autoimmune destruction of pancreatic beta cells by T cells. Interactions between both helper CD4
and cytotoxic CD8
T cells are ...essential for T1D development in NOD mice. Previous work has indicated that pathogenic T cells arise from deleterious interactions between relatively common genes which regulate aspects of T cell activation/effector function (
), peptide presentation (
,
), and T cell receptor (TCR) signaling (
). Here, we used a combination of subcongenic mapping and a CRISPR/Cas9 screen to identify the NOD-encoded mammary tumor virus (
)3 provirus as a genetic element affecting CD4
/CD8
T cell interactions through an additional mechanism, altering the TCR repertoire.
encodes a superantigen (SAg) that deletes the majority of Vβ3
thymocytes in NOD mice. Ablating
and restoring Vβ3
T cells has no effect on spontaneous T1D development in NOD mice. However, transferring
to C57BL/6 (B6) mice congenic for the NOD
MHC haplotype (B6.
) completely blocks their normal susceptibility to T1D mediated by transferred CD8
T cells transgenically expressing AI4 or NY8.3 TCRs. The entire genetic effect is manifested by Vβ3
CD4
T cells, which unless deleted by
, accumulate in insulitic lesions triggering in B6 background mice the pathogenic activation of diabetogenic CD8
T cells. Our findings provide evidence that endogenous
SAgs can influence autoimmune responses. Furthermore, since most common mouse strains have gaps in their TCR Vβ repertoire due to
, it raises questions about the role of
in other mouse models designed to reflect human immune disorders.
Neutrophils are essential for successful host eradication of bacterial pathogens and for survival to polymicrobial sepsis. During inflammation, the bone marrow provides a large reserve of neutrophils ...that are released into the peripheral circulation where they traverse to sites of infection. Although neutrophils are essential for survival, few studies have investigated the mechanisms responsible for neutrophil mobilization from the bone marrow during polymicrobial sepsis. Using a cecal ligation and puncture model of polymicrobial sepsis, we demonstrated that neutrophil mobilization from the bone marrow is not dependent on TLR4, MyD88, TRIF, IFNARα/β, or CXCR2 pathway signaling during sepsis. In contrast, we observed that bone marrow CXCL12 mRNA abundance and specific CXCL12 levels are sharply reduced, whereas splenic CXCR4 mRNA and cell surface expression are increased during sepsis. Blocking CXCL12 activity significantly reduced blood neutrophilia by inhibiting bone marrow release of granulocytes during sepsis. However, CXCL12 inhibition had no impact on the expansion of bone marrow neutrophil precursors and hematopoietic progenitors. Bone marrow neutrophil retention by CXCL12 blockade prevented blood neutrophilia, inhibited peritoneal neutrophil accumulation, allowed significant peritoneal bacterial invasion, and increased polymicrobial sepsis mortality. We concluded that changes in the pattern of CXCL12 signaling during sepsis are essential for neutrophil bone marrow mobilization and host survival but have little impact on bone marrow granulopoiesis.
Proinsulin is an abundant protein that is selectively expressed by pancreatic beta cells and has been a focus for development of antigen-specific immunotherapies for type 1 diabetes (T1D). In this ...study, we sought to comprehensively evaluate reactivity to preproinsulin by CD4 T cells originally isolated from pancreatic islets of organ donors having T1D. We analyzed 187 T cell receptor (TCR) clonotypes expressed by CD4 T cells obtained from six T1D donors and determined their response to 99 truncated preproinsulin peptide pools, in the presence of autologous B cells. We identified 14 TCR clonotypes from four out of the six donors that responded to preproinsulin peptides. Epitopes were found across all of proinsulin (insulin B-chain, C-peptide, and A-chain) including four hot spot regions containing peptides commonly targeted by TCR clonotypes derived from multiple T1D donors. Of importance, these hot spots overlap with peptide regions to which CD4 T cell responses have previously been detected in the peripheral blood of T1D patients. The 14 TCR clonotypes recognized proinsulin peptides presented by various HLA class II molecules, but there was a trend for dominant restriction with HLA-DQ, especially T1D risk alleles DQ8, DQ2, and DQ8-trans. The characteristics of the tri-molecular complex including proinsulin peptide, HLA-DQ molecule, and TCR derived from CD4 T cells in islets, provides an essential basis for developing antigen-specific biomarkers as well as immunotherapies.
Stem cell-derived β-like cells (sBC) carry the promise of providing an abundant source of insulin-producing cells for use in cell replacement therapy for patients with diabetes, potentially allowing ...widespread implementation of a practical cure. To achieve their clinical promise, sBC need to function comparably with mature adult β-cells, but as yet they display varying degrees of maturity. Indeed, detailed knowledge of the events resulting in human β-cell maturation remains obscure. Here we show that sBC spontaneously self-enrich into discreet islet-like cap structures within in vitro cultures, independent of exogenous maturation conditions. Multiple complementary assays demonstrate that this process is accompanied by functional maturation of the self-enriched sBC (seBC); however, the seBC still contain distinct subpopulations displaying different maturation levels. Interestingly, the surface protein ENTPD3 (also known as nucleoside triphosphate diphosphohydrolase-3 NDPTase3) is a specific marker of the most mature seBC population and can be used for mature seBC identification and sorting. Our results illuminate critical aspects of in vitro sBC maturation and provide important insights toward developing functionally mature sBC for diabetes cell replacement therapy.
Type 1 diabetes is an autoimmune disease in which the pancreatic β islet cells are destroyed. The main drivers of type 1 diabetes are islet-specific T cells. These cells must escape a myriad of ...tolerance mechanisms that control their activation in healthy individuals. Understanding these mechanisms is critical to developing better strategies to inhibit the disease. One pathway implicated in this process is NKG2D signaling. However, the importance of NKG2D to diabetes pathogenesis is unclear owing to conflicting results from studies in which NKG2D signaling was inhibited globally. Our previous studies indicate that these inconsistent findings are due to differential effects of NKG2D signaling in response to islet antigen and the microbiota. Therefore, new experimental approaches are required to determine the roles of NKG2D during diabetes development. To this end, we generated two novel mouse models. These are transgenic NOD mice that have enhanced NKG2D ligand expression in β cells (RIP-RAE1ε mice), and NOD mice lacking expression of the NKG2D ligand endogenously expressed in islets, H60a. We found that the RIP-RAE1ε mice and H60a-deficient mice had decreased and increased diabetes, respectively. These data demonstrate that increasing NKG2D signaling within the pancreas protects, whereas eliminating NKG2D signaling enhances, NOD diabetes. Further, we show that the NKG2D-mediated protection is associated with an increase in CD8+ central memory T cells (Tcm) in vivo, which correlates with NKG2D signaling-induced generation of both mouse and human CD8+ central memory T cells in vitro, and that CD8+ Tcm protect against diabetes in the NOD.SCID adoptive transfer model. Taken together, these data indicate that NKG2D signaling protects against diabetes by enhancing the generation of a protective CD8+ Tcm population.
Disclosure
A. Trembath: None. N. Sharma: None. I.C. Gerling: None. C.E. Mathews: None. M.A. Markiewicz: Consultant; Self; Johnson & Johnson.
Funding
American Diabetes Association (1-12-JF-41 to M.A.M.); National Institutes of Health (P30GM103326, P30CA168524, UC4DK104167, UC4DK104155, U54HD090216, P30DK020579, P30GM122731); JDRF (5-SRA-2018-557-Q-R, 17-2012-595); The Leona M. and Harry B. Helmsley Charitable Trust (2018PG-T1D053); American Association of Immunologists
Alloxan (AL)-generated Reactive Oxygen Species (ROS) selectively destroy insulin-producing pancreatic β-cells. A previous genome-wide scan (GWS) using a cohort of 296 F2 hybrids between NOD ...(AL-sensitive) and ALR (AL-resistant) mice identified linkages contributing to β-cell susceptibility or resistance to AL-induced diabetes on Chromosomes (Chr) 2, 3, 8, and a single nucleotide polymorphism in
mt-Nd2
of the mitochondrial genome (mtDNA). AL treatment of congenic and consomic NOD mouse stocks confirmed resistance linked to both the mtDNA and the Chr 8 locus from ALR NOD.mt
ALR
.ALR-(
D8Mit293-D8Mit137
). To identify possible epistatic interactions, the GWS analysis was expanded to 678 F2 mice. ALR-derived diabetes-resistance linkages on Chr 8 as well as the
mt-Nd2
a
allele were confirmed and novel additional linkages on Chr 4, 5, 6, 7, and 13 were identified. Epistasis was observed between the linkages on Chr 8 and 2 and Chr 8 and 6. Furthermore, the
mt-Nd2
genotype affected the epistatic interactions between Chr 8 and 2. These results demonstrate that a combination of nuclear-cytoplasmic genome interactions regulates β-cell sensitivity to ROS-mediated ALD.
Although B cells reactive with islet autoantigens are silenced by tolerance mechanisms in healthy individuals, they can become activated and contribute to the development of type 1 diabetes. We ...previously demonstrated that high-affinity insulin-binding B cells (IBCs) occur exclusively in the anergic (B
) compartment in peripheral blood of healthy subjects. Consistent with their activation early in disease development, high-affinity IBCs are absent from the B
compartment of some first-degree relatives (FDRs) as well as all patients with autoantibody-positive prediabetes and new-onset type 1 diabetes, a time when they are found in pancreatic islets. Loss of B
IBCs is associated with a loss of the entire B
B-cell compartment consistent with provocation by an environmental trigger or predisposing genetic factors. To investigate potential mechanisms operative in subversion of B-cell tolerance, we explored associations between HLA and non-HLA type 1 diabetes-associated risk allele genotypes and loss of B
s in FDRs. We found that high-risk HLA alleles and a subset of non-HLA risk alleles (i.e.,
rs1893217,
rs689, and
rs2872507), relevant to B- and T-cell development and function are associated with loss of anergy. Hence, the results suggest a role for risk-conferring alleles in perturbation of B-cell anergy during development of type 1 diabetes.
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