B. O. Roep

T-Cell Responses to Autoantigens in IDDM: The Search for the Holy Grail

IDDM (type I diabetes) is generally believed to result from T-cell-mediated autoimmune destruction of the insulin-producing β-cells in the pancreatic islets of Langerhans. In the last few years, considerable progress has been made with regard to the identification and characterization of candidate autoantigens recognized by autoantibodies; several of these candidate autoantigens are recognized by T-cells, including insulin, GAD65 and GAD67, heat-shock protein 65 (hsp65), and islet-cell antigen 69 (ICA69). In addition to these, a number of unidentified β-cell antigens, including insulin-secretory granule membrane proteins and a 38-kDa protein, have been shown to stimulate T-cells of IDDM patients. However, T-cell autoreactivity to islet antigens is not specific for IDDM, and the T-cell target antigens are not specific for β-cells. Moreover, the autoantigens involved in the initiation of the insulitis must be defined, and the mechanism of the T-cell-dependent β-cell destruction remains to be unraveled. This review focuses on T-cell autoreactivity in IDDM in humans and the implications of the present knowledge for immunointervention and monitoring of immunotherapeutic trials.

T-cell reactivity to 38 kD insulin-secretory-granule protein in patients with recent-onset type 1 diabetes.

Type 1 diabetes seems to be an autoimmune disease in which T cells have a substantial role. A possible target antigen was suggested by the proliferation of CD4 T cells from a newly diagnosed patient in response to a 38 kD polypeptide of the insulin-secretory-granule membrane. To see whether this reactivity is widespread at disease onset, we have generated T-cell lines in vitro from peripheral blood mononuclear cells of nineteen children of caucasoid origin with newly diagnosed type 1 diabetes and sixteen healthy controls matched for age and HLA antigens. The procedure involved two cycles of incubation with a rat beta-cell tumour subcellular fraction enriched in secretory granules and plasma membrane components, followed by a proliferation assay. Fourteen (74% [95% confidence interval 49-91%]) of the patients cell lines showed a positive proliferative response on subsequent exposure to the islet-cell antigen preparation compared with only two (13% [2-38%]) of the controls (p = 3 x 10(-4); difference 61% [44-87%]). Two subjects who had high titres of islet-cell autoantibodies (ICA) without clinical diabetes produced responsive T-cell lines. Reactivity towards the 38 kD fraction of insulin-secretory-granule membranes was found only in patients (eight of ten responders tested; 95% CI 44-98%) and one ICA-positive non-diabetic subject. Detection of an ongoing autoimmune T-cell response might be useful diagnostically and could lead to prevention of diabetes through specific immunotherapy.

Genetic Structure of IDDM1: Two Separate Regions in the Major Histocompatibility Complex Contribute to Susceptibility or Protection

We analyzed 11 markers in the IDDM1 region in 120 IDDM patients and 83 healthy control subjects who were fully matched for the highest risk HLADQA1*O3O1-DQB1*O3O2/DQA1*O5O1-DQB1*O2O1 genotype. Our study provides strong evidence that two regions in the major histocompatibility complex contribute to IDDM susceptibility or protection. First, despite selection for highest IDDM-associated risk DQ genotypes, this region displays extensive linkage disequilibrium (LD) differences between IDDM patients and control subjects. A second critical region was mapped around the microsatellite locus D6S273 centromeric of TNF, and it is ∼200 kb in size. LD analysis shows that “diabetogenic haplotypes” may have resulted from a recombination telomeric of D6S1014 in the region of D6S273 and TNFa. Haplotype analysis using HLA and microsatellite loci refines IDDM risk assessment in carriers of the HLA-DQ highest risk genotype.

Soluble forms of intercellular adhesion molecule-1 in insulin-dependent diabetes mellitus.

Soluble adhesion molecules are detectable at low levels in healthy people but are increased in various disorders. However, their physiological role is unknown. Circulating intercellular adhesion molecule-1 (cICAM-1) may modulate inflammation or arise as a consequence of inflammation. We have described elevated concentrations of cICAM-1 in subjects at risk of developing insulin-dependent diabetes mellitus (IDDM), compared with recent-onset IDDM patients and healthy controls. Here we tested the ability of a monomeric soluble recombinant form of ICAM-1 (rICAM-1), to prevent the proliferation of T cells to islet-cell and other antigens. We also tested the ability of two multivalent ICAM-1-immunoglobulin (ICAM-1-Ig) fusion proteins to stop proliferation of T cells in vitro. Autoreactive T-cell proliferation was suppressed by monoclonal antibodies directed against ICAM-1 or lymphocyte-function antigen-1 (LFA-1). Furthermore, 100 mumol rICAM-1 blocked T-cell proliferation in response to an islet-cell autoantigen, and multivalent ICAM-1-Ig fusion proteins were approximately 1,000-fold more effective. The usual interleukin-2-induced proliferation of T cells was unaffected by ICAM or ICAM-Ig. In addition, rICAM-1 blocked primary T-cell responses from peripheral blood mononuclear cells of newly diagnosed IDDM patients in concentrations similar to elevated cICAM-1 concentrations in individuals at risk for the disease. Thus, naturally circulating ICAM-1 may downregulate inflammation in subjects at risk of developing IDDM. Ig-ICAM-1 fusion proteins may thus provide novel means to intervene in the pathogenesis of autoimmune diseases.

Imogen 38: a novel 38-kD islet mitochondrial autoantigen recognized by T cells from a newly diagnosed type 1 diabetic patient.

Cell-mediated autoimmune attack directed against islet proteins of approximately 38 kD in size has been associated with type 1 diabetes. A novel murine cDNA encoding an antigen of this size was cloned using a screening procedure based on the proliferative response of a human diabetic T cell clone (1C6) to a recombinant antigen epitope library. Membrane preparations from COS 7 cells transfected with the full-length 1,267-bp cDNA elicited a proliferative response from the reporter T cells comparable to that of the defined peptide epitope and native insulinoma antigen. In vitro translation and transfection experiments suggested that the protein is initially synthesized as a 44-kD protein and then processed to the native 38-kD form through the proteolytic removal of a 54-aa NH2-terminal mitochondrial targeting sequence. Differential centrifugation, Percoll density gradient centrifugation, and immunofluorescence studies confirmed localization of the antigen to mitochondria. Northern blot, Western blot, and 1C6 T cell proliferation assays showed that, although imogen 38 was more highly expressed in beta cell than alpha cell lines, it was also present in other tissues. It is concluded that imogen 38 may be a target for bystander autoimmune attack in diabetes rather than a primary autoantigen.

Autoreactive and immunoregulatory T-cell subsets in insulin- dependent diabetes mellitus

Aims/hypothesis. Type I (insulin-dependent) diabetes mellitus is a T-cell mediated autoimmune disease. Several subsets of T-cells, in particular CD4+ and in vivo activate CD45RA+RO+ T-cells, have been shown to be increased at disease onset. The functional implications of these relative increases in CD4 T-cells were investigated. Methods. Subsets of T-cells were sorted on the basis of their activation status (CD45RA+ naïve cells, CD45RA+RO+ recently activated cells and CD45RO+ memory cells) and stimulated with autoantigens or recall antigen in vitro. Results. Proliferative responses to tetanus toxoid were primarily or exclusively observed in resting memory T-cells (CD45RO+). Autoimmune T-cell responses were, however, primarily measured in activated T-cells (CD45RA+RO+) in newly diagnosed Type I diabetic patients, whereas those with longer disease duration reacted to autoantigens with memory T-cells (CD45RO+) (p < 0.004). Interestingly, in non-diabetic control subjects not responding to autoantigens in the regular assay, considerable autoreactive T-cell responses were detectable after sorting in the CD45RO+ or CD45RA+RO+ lymphocyte subsets. Remixing these subsets showed that these autoimmune responses in activated cells could be down-modulated by CD45RA+ lymphocytes, whereas resting memory cells appeared unaffected by the suppressive CD45RA subset. Conclusion/interpretation. These results show that autoimmune T-cell responses can be linked to particular subsets which differ depending on clinical status. Furthermore, the CD45RA T-cell subset harbours lymphocytes potentially capable of suppressing autoimmune T-cell responses. The changes in responsiveness to exogenous insulin may help to unravel the mechanism by which isohormonal therapy could prevent the onset of Type I diabetes. [Diabetologia (1999) 42: 443–449]

TNFa Microsatellite Polymorphism Modulates the Risk of IDDM in Caucasians With the High-Risk Genotype HLA DQA1*0501-DQB1*0201/DQA1*0301-DQB1*0302

IDDM is a genetically controlled autoimmune disease. In particular, the loci of the HLA region on chromosome 6p are associated with the genetic risk for developing IDDM. DQAl*0501-DQBl*0201/DQAl*0301DQB1*O3O2 is the most prevalent susceptibility genotype in Caucasians, carrying a relative risk between 20 and 50 (1). However, the frequency of this high-risk genotype in the population is 10-20 times higher than the prevalence of IDDM associated with this genotype, suggesting that additional protective genes (HLA or non-HLA) and/or environmental factors can influence susceptibility to the disease (1). Recently, it has been shown that DRBl*0403 protects against IDDM in Caucasians with the high-risk heterozygous DR3-DQ2/DR4-DQ8 genotype (2), but it accounts for only -10% of the protection. Thus, DRB alleles alone cannot explain either protection or susceptibility for IDDM. To define additional markers for genetic susceptibility to or protection against IDDM in the HLA-DQ high-risk genotype group, 120 diabetic patients and 83 nondiabetic control subjects, all sharing the DQAl*0501-DQBl*0201/DQAl*0301DQBl*0302 genotype, were studied with eight microsatellite loci spanning the entire major histocompatibility complex (MHC) region and a region 2 cM telomeric of MHC. All subjects were unrelated European Caucasians and were recruited by the Belgian Diabetes Registry (2,3). HLA-typing and amplification of microsatellite markers were performed as described previously (3-5). Primer sequences for the markers HLA-F, D6S265, TNFa, D6S273, D6S1014, DQCar, TAP1, and D6S291 are available from the Genome Data Bank and were used as described previously (5). Microsatellite

An HLA-DQ alpha allele identified at DNA and protein level is strongly associated with celiac disease

An HLA-DQ alpha cDNA probe showed upon hybridization a highly significant discrepancy between the RFLP of celiac disease patients and healthy controls. The 4.0-kb Bgl II restriction fragment was present in 97% of celiac disease patients (n = 30), compared to 56% in a healthy control population (n = 72) (RR = 14.9; p less than 0.0005). At the product level all celiac disease patients tested to date have one DQ alpha chain in common, designated HLA-DQ alpha 2.3, which is associated with the 4.0-kb Bgl II fragment. This HLA-DQ alpha allele identified at the DNA level and product level seems to be a better marker for genetic susceptibility to develop celiac disease than those available to date.

β-Cell Antigen–Specific Lysis of Macrophages by CD4 T-Cell Clones From Newly Diagnosed IDDM Patient: A Putative Mechanism of T-Cell–Mediated Autoimmune Islet Cell Destruction

Immunophenotyping of the early lesion in the pancreatic islets of Langerhans demonstrates a predominance of CD4+ lymphocytes, which may be preceded by an increase in islet macrophages. This observation implies that both types of cells may be involved in autoimmune-mediated β-cell destruction leading to IDDM. In an attempt to attribute a role to β-cell antigen-specific CD4-expressing T-cell clones recently isolated from a newly diagnosed IDDM patient, we investigated whether such CD4 T-cells may be pathogenic in an in vitro cytotoxicity assay with HLA-DR-matched antigen-presenting macrophages as target. We report herein that, indeed, β-cell antigen-specific CD4+ T-cells are capable of lysing macrophages in an antigen-specific fashion. This cytotoxicity is HLA-DR restricted, T-cell receptor complex mediated, and CD4 dependent. These observations imply that both helper T-cells and macrophages may be involved in the disease process via interaction between T-cells and macrophages pulsed with β-cell antigen.

T-lymphocytes and the pathogenesis of type 1 (insulin-dependent) diabetes mellitus

In the last few years considerable progress has been made with regard to the unravelling of the processes responsible for the destruction of pancreatic P-cells in the islets of Langerhans, leading to type 1 (insulindependent) diabetes mellitus. Important B-cell autoantigens have been identified, P-cell antigen specific Tcell reactivity has been described in humans, and several non-MHC linked susceptibility loci have been mapped in the NOD mouse. Attention to genetic predisposition to type 1 diabetes in humans has moved from HLA-DR to HLA-DQ, and in particular combinations of DQ alleles of both a and B, and interest is slowly moving to non-HLA loci. The result is, that although our knowledge of the molecular basis of the disease has increased, the complexity of the genetic association and molecular processes has increased simultaneously. In this review, the latest developments in our understanding of the pathogenesis of type 1 diabetes and their implications for immunotherapy and prevention of the disease are summarized.