Tcell Receptor Characteristics And Susceptibility To

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The TCR on a given peripheral T-cell is composed of separately encoded a- and P-chains that are disulfide linked. These dimers must form a molecular complex with the multichain CD3 complex to become functionally active at the cell surface. During the entire life of an individual, T-cells undergo a maturation process that occurs primarily in the thymus. During this process, precursor stem cells, initially from the fetal liver and then from bone marrow, enter the thymic anlage, where they are induced to rearrange their germline TCRa and TCRP genes (see Fig. 6) (138). TCR gene rearrangements are essentially random, and most are nonproductive as a result of out-of-frame joints; however, these unsuccessful rearrangements are requisite for the expression of generally a single functional a/p TCR at the cell surface. Furthermore, the essentially random nature of these rearrangements among a large number of variable segments ensures an extremely large (1010-1015) repertoire of distinct antigen specificities present at the surface of the unselected thymocyte pool. Once a T-cell expresses a functional TCR at the cell surface, it is subject to either positive or negative selection events in the thymus (139,140). Both positive selection and negative selection depend on interactions among the TCR, MHC molecule, and antigenic self peptide. Positive selection occurs as thymic stromal cells bearing MHC molecules (containing self-peptide fragments) engage TCR molecules on the developing thymocytes and direct their continued maturation into functionally mature T-cells. T-cells with "useless" receptors (i.e., those that cannot bind with sufficient affinity to the MHC molecule) are not driven to mature and expand, and they eventually die. Negative selection refers to the poorly understood set of events that specifically eliminates or alternatively "anergizes" potentially autoreactive cells, thereby inducing "tolerance" to self (i.e., self-tolerance). During negative selection, factors such as affinity for self-antigen and antigen load likely influence the final outcome of cell death or clonal anergy. Thus, the peripheral T-cell repertoire of each person (including each individual of two monozy-gotic twins) is unique (141) and is a consequence of both the random generation of TCRs in the initial unselected thymocyte pool as well as of thymic positive and negative selection events.

Autoimmunity is thought to result from an imbalance between the two functionally opposite processes, tolerance induction and immune responsiveness, each dependent on the presence of class I and class II molecules with appropriate structures (dictated by the genes encoding them) that are able to present critical antigenic peptides. In genetically susceptible individuals, certain class II molecules may ineffectively present self peptides, thereby leading to inadequate negative selection of T-cell populations that could later become activated to manifest an autoimmune response. Nepom and Kwok explain the molecular basis of HLA-DQ associations with T1DM exactly on this basis (142). Paradoxically, some self peptides that normally negatively select T-cells are likely to lead to positive selection when the MHC molecule is, for example, the HLA-DQ3.2.

The HLA-DQ3.2 molecule is encoded by DQA1*0301 and DQB1*0302 genes, which are generally present on the most strongly T1DM-associated haplotype also encompassing HLA-DR4. Because of a characteristic structural motif for peptide binding, the HLA-DQ3.2 can be considered an intrinsically "unstable" MHC class II molecule. If in a

HLA-DQ*0302

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Fig. 7. Both positive and negative thymic selections contribute to form the repertoire of mature T-cells in the periphery from the precursors, or immature T-cells, originated in the bone marrow. Individuals carrying HLA-DQ alleles associated with resistance to the disease, like HLA-DQ*0602, will be able to negatively select in the thymus all the T-cells with an high affinity for peptides of the self (black dots), so that no autoreactive T-cells will be present in their peripheral blood and the chances to develop diabetes will be reduced. Individuals who have, instead, susceptibility alleles with low affinity for peptides of the self (e.g., HLA-DQ*03020) will negatively select less efficiently autoreactive T-cell clones that will then be present, even if in a relatively small number, among the peripheral T-cells. (Modified from ref. 142.)

DQ3.2-positive individual, the T-cells that are negatively selected in the thymus are only those that recognize DQ3.2-peptide complexes in a "stable" high-affinity configuration, the result can easily be the release from the thymus of mature T-cells able to establish a potentially autoimmune repertoire in the periphery. Figure 7 illustrates these concepts.

Small structural changes, then, may result in large functional changes in the antigen-presenting capabilities of the class II molecules. One might conceive that the cells from a person who is heterozygous for both DQa and DQP would contain all four chain combinations on their surface. Competition for binding the processed antigen could take place, with effective antigen binding dictated by the conformation of the antigen-binding site on each DQ dimer. As previously described, changes at either amino acid DQa-52 or DQP-57, located at opposite ends of the a-helices that form the antigen-binding groove (see Fig. 3), could alter the configuration of the groove. Changes at both positions would likely inflict a great conformational effect on the molecule's

Fig. 7. Both positive and negative thymic selections contribute to form the repertoire of mature T-cells in the periphery from the precursors, or immature T-cells, originated in the bone marrow. Individuals carrying HLA-DQ alleles associated with resistance to the disease, like HLA-DQ*0602, will be able to negatively select in the thymus all the T-cells with an high affinity for peptides of the self (black dots), so that no autoreactive T-cells will be present in their peripheral blood and the chances to develop diabetes will be reduced. Individuals who have, instead, susceptibility alleles with low affinity for peptides of the self (e.g., HLA-DQ*03020) will negatively select less efficiently autoreactive T-cell clones that will then be present, even if in a relatively small number, among the peripheral T-cells. (Modified from ref. 142.)

antigen-presentation capability. Such conformational differences may be partially responsible for the observed hierarchy in the degree of susceptibility within the group of non-Asp-57 alleles and for the differences in the degree of protection afforded by each allele within the group of Asp-57 alleles. For example, the protective effect of the Asp-57 DQB1*0502 allele prevails over that of certain susceptible alleles, such as non-Asp-57 DQB1*0501. Conversely, the susceptible allele non-Asp-57 DQB1*0302 dominates over the protective effect of Asp-57 DQB1*0301.

Competition for antigen binding would also be influenced by the relative abundance of each form of heterodimer at the cell surface, which, in turn, is likely influenced by several factors: First, certain DQa- and DQP-chains appear to be under structural constraints that limit the formation of dimers between them. For example, a-chains of the DQA1*0301 or DQA1*0501 alleles does not couple efficiently with the P-chain of the DQB1*0501 allele. Thus, persons who are heterozygous for these alleles would not be expected to readily form significant numbers of "hybrid" molecules between trans-encoded genes. Second, studies of the promoter regions of these genes suggest that the levels of transcription of the DQa- and DQP-chain genes may differ among allelic variants (136,143). These studies imply that a chain encoded by one gene may be synthesized in larger amounts than a chain encoded by the other allele, thereby increasing the probability of its participating in trans dimerization (64).

Positive- and negative-selection events can also explain genetic resistance to T1DM. In many populations, the frequency of the DQB1*0602 allele is rarely found among patients with IDDM (144,145). This suggests that this allele may play a protective role in the disease process. During thymic development, an unidentified diabetogenic peptide can preferentially bind to the DQB1*0602 molecule, and because of the relatively higher affinity and/or avidity it has with this than with other DQ molecules, it will form HLA-DQ molecule-antigenic peptide-TCR complexes more efficiently than other molecules. This could lead to negative selection and depletion of potentially self-pep-tide-reactive T-cells. Individuals with a typical DQB*0602 allele can then delete these potentially dangerous T-cells during thymic maturation and, therefore, are protected from developing diabetes (see Fig. 7). At present, carrying a "protective" DQB*0602 allele is considered as a criterion of exclusion for enrolling first-degree relatives of diabetic patients in clinical trials, such as the Diabetes Prevention Trial 1 (DPT-1), which is being carried out in the United States. This trial has been designed to prevent the progression to the clinical onset of T1DM in individuals considered at high risk for developing the disease (146). However, this does not mean that carrying the DQB1*0602 allele confers 100% protection from developing the disease (147,148). Our results suggest that prediabetics carrying the HLA haplotype DQA1*0102, DQB1*0602 have the tendency to be antibody negative for all islet autoantigens (97). Seven percent of prediabetics in this study carried the HLA haplotype DQB1*0602, which confirms previous observations that the protective effect associated with DQB 1*0602 is not absolute (147,148). We found that 40% of prediabetics carrying the HLA haplotype DQA1*0102, DQB1*0602 were African-American.

THE INSULIN GENE REGION (IDDM2)

Investigation of the insulin gene (INS) region on chromosome 11p15 as a premier candidate for genetic association with T1DM began in the early 1980s (see Fig. 8) (149-151). Insulin's central role in metabolism and blood glucose homeostasis and

MR core repeat sequence: 5' A(C/T)iGGGGT(G/C) (T/C)GGGG 3'

asesm 2200 hp (140-200 repeats} OassII 1200 bp { ? repeats) Oassl 570 bp (26-63 repeats)

INS ^ IGE2

Fig. 8. The variable number of tandem repeats (VNTR) in the 5' region of the insulin gene was grouped into three classes. The first with approx 40 repeats is called class I and the one with approx 160 repeats is class III; the number of repeats characteristic of class II genomes has not yet been formally determined. (Modified from ref. 51.)

its unique distinction as the only known P-cell-specific antigen made it a likely front-runner to account for an inherited susceptibility to diabetes. However, early studies of the human insulin gene and its relation with T1DM, T2DM, and abnormal glucose regulation were inconclusive, presumably because of the relatively small sample sizes analyzed (149-151). In 1991, Julier et al. provided evidence of genetic linkage for the insulin gene (IDDM2) with T1DM in a collection of multiplex families from France, the United States, and North Africa (152). Subsequently, the investigations of Bain et al. have confirmed the evidence of linkage between IDDM2 and type 1 diabetes (153). Importantly, Bain et al. demonstrated linkage for IDDM2 independent of the influence of HLA alleles (i.e., IDDM1) and the parental source of the IDDM2 susceptibility allele.

Detailed sequence analysis of the insulin gene region identified a polymorphic locus, which consists of a VNTR, present within the 5' regulatory region (promoter) adjacent to the coding sequence of the insulin gene. Each repeat element consists of a 14- to 15-bp DNA segment having the consensus nucleotide sequence A(C/T) AGGGGT(G/C)C(T/C/G)(G/A/T) (G/T/A)G(G/C/T) (see Fig. 8). The number of repeats within sequenced alleles ranges from 26 to >200, with 3 classes of alleles identified on the basis of overall size: class I, class II and class III. Class I INS VNTR alleles consist of 26-63 repeats, averaging 570 bp in length, and are associated with IDDM susceptibility. Class III alleles consist of 140-200 or more repeats and are considered to be protective from diabetes. In size, class III alleles are the largest variants, averaging over 2.2 kb in length. Finally, class II alleles (1.2 kb average length) are too rare in the populations studied to draw any conclusion about their association with IDDM susceptibility (154).

Detailed analyses of the insulin region indicate the presence of a number of additional polymorphism outside the VNTR region that are in strong linkage disequilibrium with the VNTR itself, which then appears to be the primary association with T1DM susceptibility. Bennet et al. reported that a 698-VNTR class I subtype is negatively associated with disease susceptibility, in contrast to other class I alleles that confer susceptibility to disease (154-156).

asesm 2200 hp (140-200 repeats} OassII 1200 bp { ? repeats) Oassl 570 bp (26-63 repeats)

Fig. 8. The variable number of tandem repeats (VNTR) in the 5' region of the insulin gene was grouped into three classes. The first with approx 40 repeats is called class I and the one with approx 160 repeats is class III; the number of repeats characteristic of class II genomes has not yet been formally determined. (Modified from ref. 51.)

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