The ways in which environmental factors participate in the progression of autoimmune diseases are not known. After initiation, it takes years before hyperglycemia develops in patients at risk for type 1 diabetes (T1D). The receptor for advanced glycation endproducts (RAGE) is a scavenger receptor of the Ig family that binds damage-associated molecular patterns and advanced glycated endproducts and can trigger cell activation. We previously found constitutive intracellular RAGE expression in lymphocytes from patients with T1D. In this article, we show that there is increased RAGE expression in T cells from at-risk euglycemic relatives who progress to T1D compared with healthy control subjects, and in the CD8+ T cells in the at-risk relatives who do versus those who do not progress to T1D. Detectable levels of the RAGE ligand high mobility group box 1 were present in serum from at-risk subjects and patients with T1D. Transcriptome analysis of RAGE+ versus RAGE− T cells from patients with T1D showed differences in signaling pathways associated with increased cell activation and survival. Additional markers for effector memory cells and inflammatory function were elevated in the RAGE+ CD8+ cells of T1D patients and at-risk relatives of patients before disease onset. These studies suggest that expression of RAGE in T cells of subjects progressing to disease predates dysglycemia. These findings imply that RAGE expression enhances the inflammatory function of T cells, and its increased levels observed in T1D patients may account for the chronic autoimmune response when damage-associated molecular patterns are released after cell injury and killing.
Receptor for advanced glycation endproducts (RAGE) is a pattern recognition receptor belonging to the Ig superfamily (1, 2). Initially described as the receptor for glycated endproducts, additional ligands belonging to the damage-associated molecular pattern (DAMP) superfamily have been identified, including high mobility group box 1 (HMGB1), S100 calcium binding proteins, amyloid fibrils, and nucleic acid backbones (3). RAGE is expressed on a wide range of cells and is thought to be involved in several pathologic conditions ranging from vascular inflammation and adaptive immune responses to Alzheimer’s disease (4–8). RAGE activation triggers downstream signaling through several inflammatory pathways, driving nuclear localization of NF-κB to promote cytokine production, cellular migration, proliferation, and survival (4, 9). However, the molecular function for RAGE in adaptive immune cells and in particular its pathologic contributions are not known.
Type 1 diabetes (T1D) is a complex autoimmune disorder resulting from failed tolerance in self-recognition. Disease progression is determined through a combination of genetic and environmental factors. Among the genetic factors, the MHC HLA genomic region is the most important modifier of disease risk (10). However, the concordance rate for diabetes among even identical twins is low, suggesting that additional environmental factors play an important role in disease progression. The link between the environmental factors and precipitation of disease has not been fully explained. Identifying these environmental factors has been challenging because the kinetics of disease are protracted and generally occur over ≥3 years (11).
T lymphocyte–mediated destruction of insulin producing pancreatic cells is thought to be the main determinant of disease progression (10). Interestingly, RAGE blockade prevents diabetes transfer by diabetogenic T cells in NOD/scid mice and delays disease recurrence in syngeneic islet transplants (12–14). We have previously shown that T cells from T1D patients express elevated levels of intracellular RAGE compared with patient T cells from other autoimmune diseases and healthy control subjects (HC) (15). However, it was unclear in those studies whether increased RAGE expression was due to hyperglycemia-induced advanced glycation endproducts accumulation or increased RAGE ligands because we also found increased levels of intracellular RAGE in T cells from patients with type 2 diabetes.
The immunologic significance of RAGE expression on T cells has not been defined. In this study, we report that intracellular RAGE expression precedes dysglycemia in at-risk relatives of patients who later progress to overt diabetes. The ligand HMGB1 augments cytokine production in activated T1D T cells. T cells from patients with T1D express elevated RAGE (Ager) mRNA levels, and the RAGE+ T cell population displays a proinflammatory gene expression profile. Interestingly, RAGE+ CD8+ T cells show enhanced survival during nutrient deprivation and associated with higher NF-κB nuclear localization. Consistent with these molecular findings, RAGE+ CD8+ T cells from patients with and at risk for T1D show higher levels of eomesodermin (EOMES) and killer cell lectin-like receptor subfamily G, member 1 (KLRG1) compared with RAGE−CD8+ T cells. Based on these findings, we suggest a mechanism whereby RAGE may link environmental events with progression of autoimmune diabetes by activating T cells that mediate cell killing.
Materials and Methods
PBMCs were obtained from 22 participants in the TrialNet TN-01, Pathway to Prevention study (Clinical Trials.gov NCT00097292). This observational study follows subjects at risk for T1D for possible enrollment in diabetes prevention trials. These subjects were designated at risk because they were relatives of a patient with T1D and found to have a positive autoantibody (anti-GAD65, anti-insulin, anti-ICA512, anti-ZnT8, or ICA). They were studied approximately every 6 mo. Of the 22 subjects, 9 did and 13 did not develop T1D over the observation period (Table I). All of the subjects had a normal hemoglobin A1c (HbA1c) levels and did not have symptoms of diabetes. The samples were obtained between June 12, 2005, and August 3, 2010. Of the 22 subjects, paired samples from 4 who did (progressors) and 4 who did not (nonprogressors) were available for flow cytometric analysis described later in this article. The at-risk population was studied over a period of 501 ± 40 d (mean ± SEM). In addition, we collected PBMCs from 23 individuals with T1D of >2 y duration and 18 HC without a personal or family history of T1D. For subjects shown in Fig. 6, paired samples were collected from four at-risk progressors and four at-risk nonprogressors 397 ± 9.6 d apart. After collection, the PBMCs were frozen (in liquid nitrogen) in our or a central laboratory. Other PBMCs were obtained from individuals with established T1D and used fresh or stored frozen in our laboratory. Institutional Review Board approval was obtained at all of the study sites, and all subjects or registered legal guardians gave written informed consent for the use of the samples.
Enriched T cells were cultured in complete RPMI 1640 media (2.05 mM l16]). After 72-h incubation at 37°C, 5% CO2 supernatants were harvested and stored at −80°C. For intracellular cytokine staining, GolgiStop (BD) was added into stimulation conditions and incubated for 6 h before staining. Cytokines were measured in the supernatants with the Cytokine Human Magnetic 11-plex Panel (Millipore) and quantified with Luminex xMAP Technology (Luminex). The effects of serum deprivation were studied in serum-free media. T cells were washed twice in 1× PBS and incubated in complete RPMI 1640 without serum for 24 h at 37°C, 5% CO2. Cells were stained for viability, cell surface and intracellular markers, and analyzed by flow cytometry. In small interfering RNA (siRNA)-transfected samples, cells were incubated in complete RPMI 1640 without serum for 48 h, stained for cell death using the LIVE/DEAD cell viability dye, and analyzed by flow cytometry.
Proximity ligation assay
Enriched T1D T cells were cultured for 4 h in 100 ng/ml His-tagged HMGB1 or His-tagged human serum albumin (HSA; Abcam), cytospun onto microscope slides, and analyzed for RAGE/HMGB1 protein interaction using Duolink in Situ Proximity Ligation Assay (PLA; Sigma) as outlined in the manufacturers’ protocols (17). Both the his-tagged HMGB1 and HSA had <1 endotoxin unit per milligram detectable LPS. In brief, mouse-RAGE (Millipore) and rabbit-6X His-tag (Sigma) directed primary Abs were added to slides and incubated in humidified chamber at room temperature for 1 h. Slides were stained with PLA probes recognizing the primary Ab species for 1 h, incubated with ligation-ligase solution for 30 min and amplification-polymerase solution for 100 min. All incubations were performed at 37°C. CD8+
CD4+ and CD8+Ager (Forward: 5′-CTGGTGCTGAAGTGTAAGGG-3′, Reverse: 5′-GAAGAGGGAGCCGTTGG-3′) or human Gapdh (Forward: 5′-ACCCACTCCTCCACCTTTGAC-3′, Reverse: 5′-TGTTGCTGTAGCCAAATTCGTT-3′) primers for quantification (Bio-Rad iQ5 Cycler).
Nanostring gene expression
T cells were isolated from PBMCs from freshly collected blood using the Pan T cell Isolation Kit (Miltenyi Biotec). RAGE+/− CD4+ and CD8+ T cells were sorted into complete RPMI 1640 media using a BD FACSAria Ilu (BD Biosciences). Cells were pelleted by centrifugation and lysed in PDK buffer (Qiagen) supplemented with 10 μl Proteinase K (Qiagen). Samples were sequentially heated at 56°C and 80°C for 12 min each, snap-frozen on liquid nitrogen, and stored at −80°C. Samples were thawed on ice and the nCounter Human Immunology v2 Codeset was used for gene expression analysis as outlined in the manufacturer’s protocol (Nanostring Technologies).
Enriched CD8+ T cells were fixed with 3% formalin for 10 min, washed, and permeabilized with 0.1% Triton X-100 + 2% FBS in PBS (no Ca/Mg) and stained with anti-p65 NF-κB Ab (Santa Cruz Biotechnology) and RAGE with anti-AGER mAb (Abnova). PE-labeled donkey Fab2 anti-rabbit IgG (Jackson Immunoresearch) and AF488-labeled goat anti-mouse IgG (H+L) (Life Technologies) were used to stain corresponding species epitopes. Cells were stained with DAPI (Sigma-Aldrich) nuclear stain for 10 min and washed twice with PBS. Nuclear localization was performed on an Amnis Imagestream-X Mark II at ×40 magnification. Nuclear localization was determined using Amnis IDEAS software (Amnis) by Pearson coefficient colocalization of DAPI and p65 NF-κB.
PBMCs were quickly thawed in water bath and incubated overnight in complete RPMI 1640 media at 37°C, 5% CO2. Enriched T cells were transfected with either human Ager
The median value for the frequency of RAGE+ CD4+ and CD8+ T cells from the at-risk subjects was calculated for each individual, using the data from all of the individual time points. Nonparametric tests (Mann–Whitney U test) were used for group and cell subset comparisons. Comparisons between RAGE+ and RAGE− measurements within each individual were made with a Wilcoxon signed-rank test. In the nanostring analysis, genes that failed to display >20 counts (natural logarithm > 3) in at least 20% of analyzed samples were determined to be below background and excluded from analysis. For each experiment, the number of individuals providing samples is indicated. All analyses were performed with GraphPad (version 6).
RAGE expression in T cells is increased in at-risk individuals who develop T1D
We analyzed RAGE expression in T cells from 22 at-risk relatives of patients with T1D who were participating in the TrialNet Pathway to Prevention Study (Table I). Multiple samples (2–5) were obtained over time from participants who did (progressors) or did not (nonprogressors) experience development of T1D over a similar observation period. All of the participants had normal HbA1c and glucose levels at the times that samples were obtained.
Representative intracellular RAGE stainings of CD4+ and CD8+ T cells are shown in Fig. 1A. For each individual, the median values describing the frequency of RAGE+ T cells among CD4+ or CD8+ T cells across all of the time points were determined and compared with the values from HCs. The median levels of RAGE expression in CD4+ and CD8+ T cells from at-risk individuals were higher than in HCs (p < 0.0001 for both; Fig. 1C). In the at-risk subjects, the median levels of RAGE expression were generally higher in progressors compared with nonprogressors to T1D in CD4+ (10.5 ± 2.62 versus 5.6 ± 0.96, p = 0.11) and significantly higher in CD8+ T cells (16.0 ± 3.47 versus 9.81 ± 2.38, p = 0.036, Mann–Whitney U test) (Fig. 1D). When all of the individual values were compared, the levels of RAGE expression were significantly higher in both CD4+ and CD8+ T cells in the progressors compared with the nonprogressors (p = 0.004 and p = 0.0004, respectively, Mann–Whitney U test) (Fig. 1E).
We also compared the changes in RAGE expression over time in the at-risk subjects. We did not detect a significant relationship between time before diagnosis of T1D and the expression of RAGE on CD4+ or CD8+ T cells (data not shown).
HMGB1/RAGE stimulation augments inflammatory cytokine release from T1D T cells
The levels of intracellular RAGE expression in T cells from the at-risk subjects were similar to levels that we had found in patients with T1D (15). Because of limited sample availability from the at-risk subjects, we carried out additional functional studies with T cells from patients after diagnosis (Table I). AGE produced with ambient hyperglycemia are not abundant in at-risk subjects in whom glucose levels are normal. We therefore tested whether other known RAGE ligands were available in the serum of patients with and at risk for T1D and found that HMGB1 was present at similar levels in the serum of at-risk progressors (4.11 ± 0.84%) and nonprogressors (4.28 ± 1.07%) as in patients with established T1D (5.33 ± 0.96%; Fig. 2A).
In previous studies we found that RAGE was expressed intracellularly, but not on the surfaces of T cells from patients with T1D, whereas RAGE expression on the cell surface of monocytes is abundant (3, 15). Because HMGB1 was found in the serum, it was unclear whether this or other RAGE ligands could interact with RAGE internally localized in T cells. To determine whether extracellular RAGE ligands could interact with intracellular RAGE, we studied RAGE/HMGB1 interactions by proximity ligation (17). Enriched T cells from T1D patients were cultured with or without his-tagged recombinant HMGB1 or HSA and stained with PLA probes detecting anti-RAGE and anti-6X his-tag. DAPI was used for nuclear visualization and CD4+ (data not shown), or CD8+ T cells were identified by counterstaining. Proximity amplification signal was observed in T1D T cells incubated overnight with 100 ng/ml recombinant HMGB1, indicating intracellular interaction between RAGE and HMGB1 (Fig. 2B).
We next tested whether HMGB1 affected functional responses of T cells from patients with T1D and HCs. Proliferation of polyclonal T cells was not affected by the addition of HMGB1 to cultures activated with anti-CD3/anti-CD28 mAbs (Supplemental Fig. 1), but there was increased cytokine production and release when HMGB1 was added to cultures from patients with T1D and HCs (p < 0.0001; Fig. 2C). Specifically, there was significantly higher release of IFN-γ (p < 0.001) and IL-6 (p < 0.05) in cultures with cells from patients with T1D versus HCs in the presence of HMGB1. Intracellular cytokine staining supported these findings as evident by the increased expression of intracellular IFN-γ in CD4+ and CD8+ T cells when HMGB1 was added to low-dose anti-CD3+ and anti-CD28+ mAbs (p < 0.01; Fig. 2D). Although not significant across multiple donors, adding HMGB1 to the cultures induced higher fold increase in IL-17a in patients with T1D versus the HCs (Fig. 2C). Interestingly, we found increased levels of IL-17a production in CD8+ T cells in the presence of HMGB1 (Fig. 2E), but not in the CD4+ T cells (data not shown).
HMGB1 may interact with several members of the TLR family (18), which are also present on T cells. To ensure the increasing proinflammatory cytokine release from T1D patients was due to RAGE, we transfected enriched T cells with Ager (RAGE) or control (Ctl) siRNA (64 ± 3.68% reduction, p = 0.0006) and stimulated them as described earlier. Reducing RAGE expression attenuated global cytokine production in activated T cells stimulated with HMGB1 (p < 0.0001). There were statistically significant reductions in IFN-γ (p = 0.04), IL-10 (p = 0.04), and IL-5 (p = 0.0002) (Fig. 2F).
Inflammatory genes and signaling pathways are constitutively elevated in RAGE+ T cells
To determine the basis for increased RAGE protein expression, we measured Ager gene expression by RT-PCR. Unstimulated CD4+ and CD8+ T cells from T1D patients displayed elevated Ager mRNA levels (2.85- and 2.71-fold, respectively) compared with HC cells (p = 0.035 and p < 0.0001, respectively; Fig. 3A, 3B). We next compared the gene expression profile between FACS-sorted RAGE+ and RAGE− T cells from patients with T1D. We used the Nanostring mRNA probe-based platform (human immunology panel) to identify differently expressed genes after fixing and permeabilization that was needed to identify RAGE by intracellular staining. RAGE+/− cells were compared within the same individual. For this exploratory analysis, gene expression was considered significantly altered if it was changed by >1.5-fold between RAGE+ and RAGE− populations with p < 0.05. Genes whose expression met these criteria in CD4+ and CD8+ T cells are shown in Fig. 3C and 3D.
We used the data on gene expression to identify differences in pathway expression between RAGE+ and RAGE− T cells with MetaCORE (Table II). Several proinflammatory signaling pathways were different in RAGE+ CD4+ and CD8+ cells, including NF-κB, IL-6, IL-12, and IL-4. In addition, pathways of cell survival and T lymphocyte proliferation/differentiation were also increased in RAGE+ T cells.
RAGE promotes CD8+ T cell survival under conditions of nutrient deprivation
One of the most highly increased pathways in the RAGE+ CD8+ T cells was prosurvival. We therefore compared the survival of RAGE+ and RAGE− T cells, from patients with T1D, under conditions of serum deprivation. When total T cells, enriched from whole PBMCs, were placed in serum-free media for 24 h, markers of cell death were reduced on RAGE+ versus RAGE− CD8+ T cell populations (11.83 ± 3.63 versus 33.50 ± 2.73%, p < 0.05; Fig. 4A, 4B). A similar trend was observed in the RAGE+ CD4+ T cells but did not reach statistical significance across multiple donors (20.42 ± 4.62 versus 35.20 ± 6.06%, p = 0.09; Supplemental Fig. 2).
To confirm the effects of RAGE on CD8+ T cell survival, we reduced RAGE expression with siRNA (38% reduction ± 4.13%, p = 0.0003), and cells were placed in serum-free media. After 48 h, the levels of cell death, detected by flow cytometry with a live/dead stain, was increased in cells transfected with Ager versus Ctl siRNA (on average by 12 ± 2.73%, p < 0.05; Fig. 4C).
Several intrinsic pathways have been proposed to determine T cell death or survival, including NF-κB that was suggested in the Nanostring analysis of the RAGE+ versus RAGE− cells. We analyzed NF-κB localization in RAGE+ and RAGE− CD8+ T cells by confocal flow cytometry. NF-κB nuclear expression was increased in five of five individuals with T1D (19 ± 2.88% higher in RAGE+ versus RAGE− cells) (p = 0.008; Fig. 4D).
RAGE+ T cells express increased markers of a proinflammatory phenotype
Consistent with our nanostring findings, in patients with T1D, there was a higher proportion of RAGE+ CD8+ T cells that were positive for EOMES and KLRG1 (Fig. 5A, 5B) and dual+ cells compared with the RAGE− CD8+ T cells (p < 0.01; Fig. 5E, 5F), suggesting a higher proportion of terminally differentiated effector memory cells. The expression of IRF4 (p < 0.01), CXCR3 (p < 0.05), and STAT3 genes (p < 0.01), found on IL-17–producing cells, were also elevated in RAGE+ CD8+ T cells (19, 20). Similar expression patterns were seen in RAGE+ versus RAGE− CD4+ T cell populations (Supplemental Fig. 3A).
We also analyzed the phenotypes of T cells from at-risk subjects who progressed to T1D using samples from two separate study visits ∼1 y apart (Fig. 6). Similar to our findings in patients with T1D, we found that the RAGE+ cells expressed higher levels of EOMES and KLRG1 in CD8+ T cells (p = 0.0016 and 0.03, respectively; Fig. 6A, 6B). The expression of IRF4 also tended to be increased on the RAGE+ versus RAGE− CD8+ T cells (p = 0.09, by two-way ANOVA), but we did not detect a significant difference in the level of CXCR3 expression (p = 0.16; Fig. 6C, 6D). There were similar findings among CD4+ T cells (Supplemental Fig. 4). We did not detect changes in the frequency of the EOMES, KLRG1, or dual+ cells between the two study visits before diabetes onset (Fig. 6).
After the initiation of autoimmunity, coinciding with the appearance of autoantibodies, it takes years for T1D to occur. Studies from our group and others have suggested that the pathologic process is relatively quiet until the peridiagnosis period when there is a climactic increase in cell killing and failure in insulin secretion (21). Our studies of RAGE expression and function in T cells from at-risk subjects and patients with T1D suggest a mechanism that may account for the persistence of chronic inflammation and the accelerated pathology in the peridiagnosis period. We found high levels of RAGE expression in CD4+ and CD8+ T cells of autoantibody-positive, at-risk subjects who later progressed to disease before the development of dysglycemia or overt diabetes compared with HCs or at-risk nonprogressors. Gene expression in RAGE+ cells suggested activated inflammatory pathways, confirmed by higher levels of pathologic cytokine production when T cells from patients with T1D were activated with the RAGE ligand HMGB1 compared with T cells from HC. We found that the RAGE ligand, HMGB1, is able to gain entry into T cells and bind with the internally expressed receptor. RAGE expression was associated with increased T cell survival. Thus, the expression of RAGE in T1D patient T cells and its functional activity may account for the persistence of underlying inflammation and RAGE+ T cell pathologic function when DAMPs are released after cell injury and killing, or when glycated endproducts are available by transient elevations of glucose levels. Clinical data showing a rapid decline in cell function in the peridiagnosis period compared with earlier periods are consistent with this proposed mechanism (21, 22).
RAGE expression was higher in T cells from individuals who progressed to overt T1D compared with nonprogressors. We do not know the basis for the persistence of RAGE expression on these T cells. RAGE ligands can increase RAGE expression, but these subjects did not have overt hyperglycemia that would increase glycated endproducts. We cannot exclude the possibility that there are periodic excursions of glucose levels that generate AGEs, but in addition to normal oral glucose tolerance tests, another measure of hyperglycemia, HbA1c, was also normal in our at-risk subjects. Likewise, continuous exposure to Ags may be involved in the maintained expression, but the relatively high frequency of RAGE+ cells is unlikely to be accounted for exclusively by cells that are specific for diabetes-associated Ags. We did not identify a change in the frequency of RAGE+ CD4+ or CD8+ T cells over time in the at-risk subjects followed to overt T1D. However, the number of subjects that we studied was limited and the sampling interval was large. A more detailed time course of the kinetics of RAGE expression would allow us to address this question further.
Functional and phenotypic features of RAGE+ T cells suggest a role of these cells in diabetogenesis where RAGE ligands are available. RAGE+ CD8+ T cells expressed CXCR3 and EOMES and a high level of EOMES/KLRG1 dual positivity in patients with T1D, suggesting differentiated memory Th1 or Tc1 cells. The gene expression and NF-κB localization findings indicate that RAGE+ T cells exhibit a predisposed inflammatory phenotype, and when activated in the presence of HMGB1, the cells produced higher levels of IFN-γ, an important cytokine released by pathologic T cells in T1D (23). Furthermore, as a result of high levels of CXCR3 expression, the RAGE+ cells may traffic to the islets in response to IP-10 (CXCL10) because IP-10 is abundantly expressed in the pancreatic islets and is found in the serum of newly diagnosed patients with diabetes and at-risk individuals (24). We also found increased expression of the Th17/Tc17 cell lineage markers IRF4 and STAT3 in the RAGE+ CD8+ T cells in patients with T1D. However, we did not find a significant difference in either IRF4 or CXCR3 expression in the RAGE+ versus RAGE− CD8+ T cells from the at-risk progressors that may suggest that RAGE expression occurs before terminal differentiation of the T cells. However, we did find increased frequency of EOMES and KLRG1+ cells in the RAGE+ versus RAGE− CD8+ T cells from the at-risk progressors at both study visits. Further studies of the relationships between RAGE expression and activation and T cell differentiation are needed to resolve the differences in the at-risk subjects and patients. In our previous studies we found increased IL-17A production by T cells after activation with PMA/ionomycin (15), but we did not identify statistically significant differences upon HMGB1 stimulation. There are several possible explanations for this discrepancy including differences in the cell populations studied and methods of activation.
In the endosomes, RAGE may remove DAMPs that are generated within T cells by cellular damage. In this way, intracellular RAGE expression may improve cell survival because in serum-deprived media, RAGE+ cells showed improved survival that was lessened by RAGE siRNA. Previous work demonstrates that the NF-κB family of transcription factors is critical for T cell survival, and RAGE+ T cells constitutively express high levels of nuclear NF-κB (25). Additional genes (Relb) and signaling pathways (p53) that were activated in RAGE+ T cells have been shown to protect various types of cancer cells from nutrient-deprived apoptosis (26, 27). A role for these mechanisms in terms of RAGE-mediated survival of pathologic T cells in T1D requires further evaluation.
Our study has limitations that are important to recognize. Our sample size of at-risk subjects was relatively small, but they were studied on multiple occasions. Furthermore, we followed the nonprogressors only for the same period as the progressors; therefore, we cannot conclude that the nonprogressors are protected from disease. Our studies used HMGB1 as the model RAGE ligand, but several others have been described (1). HMGB1 is a well-characterized ligand that we show is present in the serum of patients with diabetes, those at risk, and even in HCs (Fig. 2A). Although other ligands (e.g., glycosylated proteins) may be important in the disease mechanism, our studies with this widely found ligand enabled us to focus on the significance of RAGE expression on T cells. We did not study the effects of RAGE ligands on other immune and nonimmune cells that may indirectly affect T cells. Although we have taken measures to limit non–T cell contributions within our experiments, we cannot rule out the indirect effects of other immune cells in vivo. Finally, our data showing increased Ager expression in the T cells from patients with T1D, as well as our siRNA studies, suggest that RAGE is made by the T cells themselves. However, we cannot exclude the possibility that RAGE is taken up into endosomes from T cells where it may still have effects on the T cells that we describe.
In summary, these studies suggest that intracellular RAGE expression may identify at-risk individuals who progress to T1D. They also suggest mechanisms whereby environmental factors may accelerate T1D progression by activating RAGE+ T cells. Even nonspecific mediators may trigger the cells that cause disease, explaining the clinical course and highlighting the difficulties in identifying single environmental factors that cause the disease.
TrialNet Study Group
Steering committee: C. Greenbaum (Benaroya Research Institute), M. Anderson (University of California, San Francisco), P. Antinozzi (Wake Forest University), M. Atkinson (University of Florida), M. Battaglia (San Raffaele University), D. Becker (University of Pittsburgh), P. Bingley (University of Bristol), E. Bosi (San Raffaele University), J. Buckner (Benaroya Research Institute), Mark Clements (Children’s Mercy Hospital), P. Colman (Walter & Eliza Hall Institute of Medical Research), L. DiMeglio (Indiana University), S. Gitelman (University of California, San Francisco), R. Goland (Columbia University), P. Gottlieb (Barbara Davis Center for Childhood Diabetes), K. Herold (Yale University), R. Insel (Juvenile Diabetes Research Foundation), T. Kay (St. Vincent’s Institute of Medical Research), M. Knip (University of Helsinki), J. Krischer (University of South Florida), A. Lernmark (Skane University), J.B. Marks (University of Miami), W. Moore (Children’s Mercy Hospital), A. Moran (University of Minnesota), Andrew Muir (Emory University), J. Palmer (University of Washington), M. Peakman (King’s College), L. Philipson (University of Chicago), A. Pugliese (University of Miami), P. Raskin (University of Texas Southwestern), M. Redondo (Baylor University), H. Rodriguez (University of South Florida), B. Roep (Leiden University Medical Center), W. Russell (Vanderbilt University), L. Spain (National Institute of Diabetes and Digestive and Kidney Diseases [NIDDK]), D.A. Schatz (University of Florida), J. Sosenko (University of Miami), D. Wherrett (University of Toronto), D. Wilson (Stanford University), W. Winter (University of Florida), A. Ziegler (Forschergruppe Diabetes).
Previous members: J.S. Skyler (University of Miami) (Chair), C. Benoist (Joslin Diabetes Center), J. Blum (Indiana University), K. Bourcier, P. Chase (Barbara Davis Center for Childhood Diabetes), M. Clare-Salzler (University of Florida), R. Clynes (Columbia University), G. Eisenbarth (Barbara Davis Center for Childhood Diabetes), C.G. Fathman (Stanford University), G. Grave (National Institute of Child Health and Human Development), B. Hering (University of Minnesota), F. Kaufman (Children’s Hospital Los Angeles), E. Leschek (NIDDK), J. Mahon (University of Western Ontario), K. Nanto-Salonen University of Turku), G. Nepom (Benaroya Research Institute), T. Orban (Joslin Diabetes Center), R. Parkman (Children’s Hospital Los Angeles), M. Pescovitz (Indiana University), J. Peyman (National Institute of Allergy and Infectious Disease), M. Roncarolo (San Raffaele University), P. Savage (NIDDK), O. Simell (University of Turku), R. Sherwin (Yale University), M. Siegelman (University of Texas Southwestern), A. Steck (Barbara Davis Center for Childhood Diabetes), J. Thomas (Vanderbilt University), M. Trucco (University of Pittsburgh), J. Wagner (University of Minnesota).
The authors have no financial conflicts of interest.
We thank the following individuals for contributions: L. Devine and C. Wang (Yale Department of Laboratory Medicine) for help in FACS and review of the manuscript, O.I. Henegariu (Yale School of Medicine) for anti-human RAGE fluorophore conjugation, and J. Esko (University of California, San Diego) for providing recombinant HMGB1.
↵1 All authors and their affiliations appear at the end of this article.
This work was supported by National Institutes of Health Grants R01 DK057846, U01 AI102011-04, and U01 DK085466 and Juvenile Diabetes Research Foundation Grant SRA 2014-158. The Type 1 Diabetes TrialNet Pathway to Prevention Study Group is a clinical trials network supported by the National Institutes of Health through the National Institute of Diabetes and Digestive and Kidney Diseases, the National Institute of Allergy and Infectious Diseases, and The Eunice Kennedy Shriver National Institute of Child Health and Human Development, per cooperative agreements.
The online version of this article contains supplemental material.
Abbreviations used in this article:
- damage-associated molecular pattern
- hemoglobin A1c
- healthy control subject
- high mobility group box 1
- human serum albumin
- IFN regulatory factor-4
- killer cell lectin-like receptor subfamily G, member 1
- National Institute of Diabetes and Digestive and Kidney Diseases
- proximity ligation assay
- receptor for advanced glycation endproducts
- small interfering RNA
- type 1 diabetes.
- Received February 5, 2016.
- Accepted August 18, 2016.
- Copyright © 2016 by The American Association of Immunologists, Inc.