Background Type 1 diabetes (T1D) is a T-cell mediated autoimmune disease targeting the insulin-producing pancreatic β cells. Naturally occurring FOXP3+CD4+CD25high regulatory T cells (Tregs) play an important role in dominant tolerance, suppressing autoreactive CD4+ effector T cell activity. Previously, in both recent-onset T1D patients and β cell antibody-positive at-risk individuals, we observed increased apoptosis and decreased function of polyclonal Tregs in the periphery. Our objective here was to elucidate the genes and signaling pathways triggering apoptosis in Tregs from T1D subjects. Principal Findings Gene expression profiles of unstimulated Tregs from recent-onset T1D (n = 12) and healthy control subjects (n = 15) were generated. Statistical analysis was performed using a Bayesian approach that is highly efficient in determining differentially expressed genes with low number of replicate samples in each of the two phenotypic groups. Microarray analysis showed that several cytokine/chemokine receptor genes, HLA genes, GIMAP family genes and cell adhesion genes were downregulated in Tregs from T1D subjects, relative to control subjects. Several downstream target genes of the AKT and p53 pathways were also upregulated in T1D subjects, relative to controls. Further, expression signatures and increased apoptosis in Tregs from T1D subjects partially mirrored the response of healthy Tregs under conditions of IL-2 deprivation. CD4+ effector T-cells from T1D subjects showed a marked reduction in IL-2 secretion. This could indicate that prior to and during the onset of disease, Tregs in T1D may be caught up in a relatively deficient cytokine milieu. Conclusions In summary, expression signatures in Tregs from T1D subjects reflect a cellular response that leads to increased sensitivity to apoptosis, partially due to cytokine deprivation. Further characterization of these signaling cascades should enable the detection of genes that can be targeted for restoring Treg function in subjects predisposed to T1D.
Donath MY, Storling J, Maedler K, Mandrup-Poulsen T (2003) Inflammatory mediators and islet beta-cell failure: a link between type 1 and type 2 diabetes. J Mol Med 81: 455–470.
[3]
Bacchetta R, Gambineri E, Roncarolo MG (2007) Role of regulatory T cells and FOXP3 in human diseases. J Allergy Clin Immunol 120: 227–235; quiz 236–227.
[4]
Putnam AL, Vendrame F, Dotta F, Gottlieb PA (2005) CD4+CD25high regulatory T cells in human autoimmune diabetes. J Autoimmun 24: 55–62.
[5]
Kukreja A, Cost G, Marker J, Zhang C, Sun Z, et al. (2002) Multiple immuno-regulatory defects in type-1 diabetes. J Clin Invest 109: 131–140.
[6]
Lindley S, Dayan CM, Bishop A, Roep BO, Peakman M, et al. (2005) Defective suppressor function in CD4(+)CD25(+) T-cells from patients with type 1 diabetes. Diabetes 54: 92–99.
[7]
Brusko TM, Wasserfall CH, Clare-Salzler MJ, Schatz DA, Atkinson MA (2005) Functional defects and the influence of age on the frequency of CD4+ CD25+ T-cells in type 1 diabetes. Diabetes 54: 1407–1414.
[8]
Abdul-Rasoul M, Habib H, Al-Khouly M (2006) ‘The honeymoon phase’ in children with type 1 diabetes mellitus: frequency, duration, and influential factors. Pediatr Diabetes 7: 101–107.
[9]
Bonfanti R, Bognetti E, Meschi F, Brunelli A, Riva MC, et al. (1998) Residual beta-cell function and spontaneous clinical remission in type 1 diabetes mellitus: the role of puberty. Acta Diabetol 35: 91–95.
[10]
Glisic-Milosavljevic S, Waukau J, Jailwala P, Jana S, Khoo HJ, et al. (2007) At-risk and recent-onset type 1 diabetic subjects have increased apoptosis in the CD4+CD25+ T-cell fraction. PLoS ONE 2: e146.
[11]
Knoechel B, Lohr J, Zhu S, Wong L, Hu D, et al. (2006) Functional and molecular comparison of anergic and regulatory T lymphocytes. J Immunol 176: 6473–6483.
[12]
Pfoertner S, Jeron A, Probst-Kepper M, Guzman CA, Hansen W, et al. (2006) Signatures of human regulatory T cells: an encounter with old friends and new players. Genome Biol 7: R54.
[13]
Sugimoto N, Oida T, Hirota K, Nakamura K, Nomura T, et al. (2006) Foxp3-dependent and -independent molecules specific for CD25+CD4+ natural regulatory T cells revealed by DNA microarray analysis. Int Immunol 18: 1197–1209.
[14]
Fontenot JD, Gavin MA, Rudensky AY (2003) Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat Immunol 4: 330–336.
[15]
Gavin MA, Clarke SR, Negrou E, Gallegos A, Rudensky A (2002) Homeostasis and anergy of CD4(+)CD25(+) suppressor T cells in vivo. Nat Immunol 3: 33–41.
[16]
Anderson PO, Manzo BA, Sundstedt A, Minaee S, Symonds A, et al. (2006) Persistent antigenic stimulation alters the transcription program in T cells, resulting in antigen-specific tolerance. Eur J Immunol 36: 1374–1385.
[17]
Hill JA, Feuerer M, Tash K, Haxhinasto S, Perez J, et al. (2007) Foxp3 transcription-factor-dependent and -independent regulation of the regulatory T cell transcriptional signature. Immunity 27: 786–800.
[18]
Herman AE, Freeman GJ, Mathis D, Benoist C (2004) CD4+CD25+ T regulatory cells dependent on ICOS promote regulation of effector cells in the prediabetic lesion. J Exp Med 199: 1479–1489.
[19]
Chen Z, Herman AE, Matos M, Mathis D, Benoist C (2005) Where CD4+CD25+ T reg cells impinge on autoimmune diabetes. J Exp Med 202: 1387–1397.
[20]
Kaizer EC, Glaser CL, Chaussabel D, Banchereau J, Pascual V, et al. (2007) Gene Expression in Peripheral Blood Mononuclear Cells from Children with Diabetes. J Clin Endocrinol Metab.
[21]
Orban T, Kis J, Szereday L, Engelmann P, Farkas K, et al. (2007) Reduced CD4+ T-cell-specific gene expression in human type 1 diabetes mellitus. J Autoimmun 28: 177–187.
[22]
Furtado GC, Curotto de Lafaille MA, Kutchukhidze N, Lafaille JJ (2002) Interleukin 2 signaling is required for CD4(+) regulatory T cell function. J Exp Med 196: 851–857.
[23]
Setoguchi R, Hori S, Takahashi T, Sakaguchi S (2005) Homeostatic maintenance of natural Foxp3(+) CD25(+) CD4(+) regulatory T cells by interleukin (IL)-2 and induction of autoimmune disease by IL-2 neutralization. J Exp Med 201: 723–735.
[24]
Wolf M, Schimpl A, Hunig T (2001) Control of T cell hyperactivation in IL-2-deficient mice by CD4(+)CD25(-) and CD4(+)CD25(+) T cells: evidence for two distinct regulatory mechanisms. Eur J Immunol 31: 1637–1645.
[25]
Almeida AR, Legrand N, Papiernik M, Freitas AA (2002) Homeostasis of peripheral CD4+ T cells: IL-2R alpha and IL-2 shape a population of regulatory cells that controls CD4+ T cell numbers. J Immunol 169: 4850–4860.
[26]
Malek TR, Yu A, Vincek V, Scibelli P, Kong L (2002) CD4 regulatory T cells prevent lethal autoimmunity in IL-2Rbeta-deficient mice. Implications for the nonredundant function of IL-2. Immunity 17: 167–178.
[27]
Thornton AM, Donovan EE, Piccirillo CA, Shevach EM (2004) Cutting edge: IL-2 is critically required for the in vitro activation of CD4+CD25+ T cell suppressor function. J Immunol 172: 6519–6523.
[28]
de la Rosa M, Rutz S, Dorninger H, Scheffold A (2004) Interleukin-2 is essential for CD4+CD25+ regulatory T cell function. Eur J Immunol 34: 2480–2488.
[29]
Bayer AL, Yu A, Adeegbe D, Malek TR (2005) Essential role for interleukin-2 for CD4(+)CD25(+) T regulatory cell development during the neonatal period. J Exp Med 201: 769–777.
[30]
Tang Q, Adams JY, Penaranda C, Melli K, Piaggio E, et al. (2008) Central role of defective interleukin-2 production in the triggering of islet autoimmune destruction. Immunity 28: 687–697.
[31]
Yamanouchi J, Rainbow D, Serra P, Howlett S, Hunter K, et al. (2007) Interleukin-2 gene variation impairs regulatory T cell function and causes autoimmunity. Nat Genet 39: 329–337.
[32]
Vukmanovic-Stejic M, Zhang Y, Cook JE, Fletcher JM, McQuaid A, et al. (2006) Human CD4+ CD25hi Foxp3+ regulatory T cells are derived by rapid turnover of memory populations in vivo. J Clin Invest 116: 2423–2433.
[33]
Aswad F, Kawamura H, Dennert G (2005) High sensitivity of CD4+CD25+ regulatory T cells to extracellular metabolites nicotinamide adenine dinucleotide and ATP: a role for P2X7 receptors. J Immunol 175: 3075–3083.
[34]
Mootha VK, Lindgren CM, Eriksson KF, Subramanian A, Sihag S, et al. (2003) PGC-1alpha-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes. Nat Genet 34: 267–273.
[35]
Hosack DA, Dennis G Jr, Sherman BT, Lane HC, Lempicki RA (2003) Identifying biological themes within lists of genes with EASE. Genome Biol 4: R70.
[36]
Nakamoto Y, Kaneko S, Kobayashi K (2002) Increased susceptibility to apoptosis and attenuated Bcl-2 expression in T lymphocytes and monocytes from patients with advanced chronic hepatitis C. J Leukoc Biol 72: 49–55.
[37]
Chalah A, Khosravi-Far R (2008) The mitochondrial death pathway. Adv Exp Med Biol 615: 25–45.
[38]
Dalberg U, Markholst H, Hornum L (2007) Both Gimap5 and the diabetogenic BBDP allele of Gimap5 induce apoptosis in T cells. Int Immunol 19: 447–453.
[39]
Nitta T, Nasreen M, Seike T, Goji A, Ohigashi I, et al. (2006) IAN family critically regulates survival and development of T lymphocytes. PLoS Biol 4: e103.
[40]
Dion C, Carter C, Hepburn L, Coadwell WJ, Morgan G, et al. (2005) Expression of the Ian family of putative GTPases during T cell development and description of an Ian with three sets of GTP/GDP-binding motifs. Int Immunol 17: 1257–1268.
[41]
Ward SG, Westwick J (1998) Chemokines: understanding their role in T-lymphocyte biology. Biochem J 333 (Pt3): 457–470.
[42]
Palacios R (1982) Mechanism of T cell activation: role and functional relationship of HLA-DR antigens and interleukins. Immunol Rev 63: 73–110.
[43]
Papiernik M, de Moraes ML, Pontoux C, Vasseur F, Penit C (1998) Regulatory CD4 T cells: expression of IL-2R alpha chain, resistance to clonal deletion and IL-2 dependency. Int Immunol 10: 371–378.
[44]
Rabinovitch A, Suarez-Pinzon WL (2007) Roles of cytokines in the pathogenesis and therapy of type 1 diabetes. Cell Biochem Biophys 48: 159–163.
[45]
Fleischer A, Duhamel M, Lopez-Fernandez LA, Munoz M, Rebollo MP, et al. (2007) Cascade of transcriptional induction and repression during IL-2 deprivation-induced apoptosis. Immunol Lett 112: 9–29.
[46]
Kaye WA, Adri MN, Soeldner JS, Rabinowe SL, Kaldany A, et al. (1986) Acquired defect in interleukin-2 production in patients with type I diabetes mellitus. N Engl J Med 315: 920–924.
[47]
Giordano C, Panto F, Caruso C, Modica MA, Zambito AM, et al. (1989) Interleukin 2 and soluble interleukin 2-receptor secretion defect in vitro in newly diagnosed type I diabetic patients. Diabetes 38: 310–315.
[48]
Bosque A, Marzo I, Naval J, Anel A (2007) Apoptosis by IL-2 deprivation in human CD8+ T cell blasts predominates over death receptor ligation, requires Bim expression and is associated with Mcl-1 loss. Mol Immunol 44: 1446–1453.
[49]
Devireddy LR, Green MR (2003) Transcriptional program of apoptosis induction following interleukin 2 deprivation: identification of RC3, a calcium/calmodulin binding protein, as a novel proapoptotic factor. Mol Cell Biol 23: 4532–4541.
[50]
Stahl M, Dijkers PF, Kops GJ, Lens SM, Coffer PJ, et al. (2002) The forkhead transcription factor FoxO regulates transcription of p27Kip1 and Bim in response to IL-2. J Immunol 168: 5024–5031.
[51]
Ahmed NN, Grimes HL, Bellacosa A, Chan TO, Tsichlis PN (1997) Transduction of interleukin-2 antiapoptotic and proliferative signals via Akt protein kinase. Proc Natl Acad Sci U S A 94: 3627–3632.
[52]
You H, Pellegrini M, Tsuchihara K, Yamamoto K, Hacker G, et al. (2006) FOXO3a-dependent regulation of Puma in response to cytokine/growth factor withdrawal. J Exp Med 203: 1657–1663.
[53]
You H, Yamamoto K, Mak TW (2006) Regulation of transactivation-independent proapoptotic activity of p53 by FOXO3a. Proc Natl Acad Sci U S A 103: 9051–9056.
[54]
Tran H, Brunet A, Grenier JM, Datta SR, Fornace AJ Jr, et al. (2002) DNA repair pathway stimulated by the forkhead transcription factor FOXO3a through the Gadd45 protein. Science 296: 530–534.
[55]
Furukawa-Hibi Y, Kobayashi Y, Chen C, Motoyama N (2005) FOXO transcription factors in cell-cycle regulation and the response to oxidative stress. Antioxid Redox Signal 7: 752–760.
[56]
Faustman D, Li XP, Lin HY, Fu YE, Eisenbarth G, et al. (1991) Linkage of faulty major histocompatibility complex class I to autoimmune diabetes. Science 254: 1756–1761.
[57]
Hao W, Gladstone P, Engardt S, Greenbaum C, Palmer JP (1996) Major histocompatibility complex class I molecule expression is normal on peripheral blood lymphocytes from patients with insulin-dependent diabetes mellitus. J Clin Invest 98: 1613–1618.
[58]
Anal O, Akkoc N, Sen A, Yesil S, Yuksel F, et al. (1997) MHC class I antigen expression in patients with IDDM and their siblings. J Pediatr Endocrinol Metab 10: 391–394.
[59]
Baecher-Allan C, Wolf E, Hafler DA (2006) MHC class II expression identifies functionally distinct human regulatory T cells. J Immunol 176: 4622–4631.
[60]
Nitta T, Takahama Y (2007) The lymphocyte guard-IANs: regulation of lymphocyte survival by IAN/GIMAP family proteins. Trends Immunol 28: 58–65.
[61]
Kupfer R, Lang J, Williams-Skipp C, Nelson M, Bellgrau D, et al. (2007) Loss of a gimap/ian gene leads to activation of NF-kappaB through a MAPK-dependent pathway. Mol Immunol 44: 479–487.
[62]
MacMurray AJ, Moralejo DH, Kwitek AE, Rutledge EA, Van Yserloo B, et al. (2002) Lymphopenia in the BB rat model of type 1 diabetes is due to a mutation in a novel immune-associated nucleotide (Ian)-related gene. Genome Res 12: 1029–1039.
[63]
Hornum L, Romer J, Markholst H (2002) The diabetes-prone BB rat carries a frameshift mutation in Ian4, a positional candidate of Iddm1. Diabetes 51: 1972–1979.
[64]
Marson A, Kretschmer K, Frampton GM, Jacobsen ES, Polansky JK, et al. (2007) Foxp3 occupancy and regulation of key target genes during T-cell stimulation. Nature 445: 931–935.
[65]
Glisic-Milosavljevic S, Wang T, Koppen M, Kramer J, Ehlenbach S, et al. (2007) Dynamic changes in CD4+ CD25+(high) T cell apoptosis after the diagnosis of type 1 diabetes. Clin Exp Immunol 150: 75–82.
[66]
Alberti KG, Zimmet PZ (1998) Definition, diagnosis and classification of diabetes mellitus and its complications. Part 1: diagnosis and classification of diabetes mellitus provisional report of a WHO consultation. Diabet Med 15: 539–553.
[67]
Piccirillo CA, Letterio JJ, Thornton AM, McHugh RS, Mamura M, et al. (2002) CD4(+)CD25(+) regulatory T cells can mediate suppressor function in the absence of transforming growth factor beta1 production and responsiveness. J Exp Med 196: 237–246.
[68]
Jonuleit H, Schmitt E, Kakirman H, Stassen M, Knop J, et al. (2002) Infectious tolerance: human CD25(+) regulatory T cells convey suppressor activity to conventional CD4(+) T helper cells. J Exp Med 196: 255–260.
[69]
Baecher-Allan C, Viglietta V, Hafler DA (2004) Human CD4+CD25+ regulatory T cells. Semin Immunol 16: 89–98.
[70]
Hartigan-O'Connor DJ, Poon C, Sinclair E, McCune JM (2007) Human CD4+ regulatory T cells express lower levels of the IL-7 receptor alpha chain (CD127), allowing consistent identification and sorting of live cells. J Immunol Methods 319: 41–52.
[71]
Liu W, Putnam AL, Xu-Yu Z, Szot GL, Lee MR, et al. (2006) CD127 expression inversely correlates with FoxP3 and suppressive function of human CD4+ T reg cells. J Exp Med 203: 1701–1711.
[72]
Gentleman RC, Carey VJ, Bates DM, Bolstad B, Dettling M, et al. (2004) Bioconductor: open software development for computational biology and bioinformatics. Genome Biol 5: R80.
[73]
Tusher VG, Tibshirani R, Chu G (2001) Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci U S A 98: 5116–5121.
[74]
Baldi P, Long AD (2001) A Bayesian framework for the analysis of microarray expression data: regularized t -test and statistical inferences of gene changes. Bioinformatics 17: 509–519.
[75]
Smyth GK (2004) Linear models and empirical bayes methods for assessing differential expression in microarray experiments. Stat Appl Genet Mol Biol 3: Article3.
[76]
Turro E, Bochkina N, Hein AM, Richardson S (2007) BGX: a Bioconductor package for the Bayesian integrated analysis of Affymetrix GeneChips. BMC Bioinformatics Under Review.
[77]
Hein AM, Richardson S, Causton HC, Ambler GK, Green PJ (2005) BGX: a fully Bayesian integrated approach to the analysis of Affymetrix GeneChip data. Biostatistics 6: 349–373.
[78]
Hein AM, Richardson S (2006) A powerful method for detecting differentially expressed genes from GeneChip arrays that does not require replicates. BMC Bioinformatics 7: 353.
[79]
Efron B (2003) Large-scale simultaneous hypothesis testing: the choice of a null hypothesis. J Am Statist Assoc: 96: 99.
[80]
Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, et al. (2000) Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat Genet 25: 25–29.
[81]
Glisic-Milosavljevic S, Waukau J, Jana S, Jailwala P, Rovensky J, et al. (2005) Comparison of apoptosis and mortality measurements in peripheral blood mononuclear cells (PBMCs) using multiple methods. Cell Prolif 38: 301–311.
