NATURE | VOL 406 | 17 AUGUST 2000 | www.nature.com 739 letters to nature ................................................................. Progression of autoimmune diabetes driven by avidity maturation of a T-cell population Abdelaziz Amrani*, Joan Verdaguer*, Pau Serra*, Sabrina Tafuro², Rusung Tan³ & Pere Santamaria* * Department of Microbiology and Infectious Diseases, Faculty of Medicine, University of Calgary, Health Sciences Centre, 3330 Hospital Drive NW, Calgary, Alberta T2N 4N1, Canada ² MRC Human Immunology Unit, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9DU, UK ³ Department of Pathology and Laboratory Medicine, University of British Columbia and BC's Children's Hospital, 4480 Oak Street, Vancouver, V6H 3V4, British Columbia, Canada .............................................................................................................................................. For unknown reasons, autoimmune diseases such as type 1 diabetes develop after prolonged periods of in¯ammation of mononuclear cells in target tissues 1 . Here we show that progres- sion of pancreatic islet in¯ammation to overt diabetes in non- obese diabetic (NOD) mice is driven by the `avidity maturation' of a prevailing, pancreatic beta-cell-speci®c T-lymphocyte popula- tion carrying the CD8 antigen. This T-lymphocyte population recognizes two related peptides (NRP and NRP-A7) 2 in the context of H-2K d class I molecules of the major histocompatibility com- plex (MHC). As pre-diabetic NOD mice age, their islet-associated CD8 + T lymphocytes contain increasing numbers of NRP-A7- reactive cells, and these cells bind NRP-A7/H-2K d tetramers with increased speci®city, increased avidity and longer half-lives. Repeated treatment of pre-diabetic NOD mice with soluble NRP-A7 peptide blunts the avidity maturation of the NRP-A7- reactive CD8 + T-cell population by selectively deleting those clonotypes expressing T-cell receptors with the highest af®nity and lowest dissociation rates for peptide±MHC binding. This inhibits the local production of T cells that are cytotoxic to beta cells, and halts the progression from severe insulitis to diabetes. We conclude that avidity maturation of pathogenic T-cell popula- tions may be the key event in the progression of benign in¯am- mation to overt disease in autoimmunity. Nonobese diabetic mice develop in¯ammation of pancreatic islets (insulitis) at 3 weeks of age, but do not begin to develop diabetes until 10 weeks later 1 . Although CD8 + cytotoxic T lympho- cytes (CTLs) are well established as killers of insulin-producing pancreatic beta cells in autoimmune diabetes 3±9 , the mechanisms that drive the progression of insulitis to overt diabetes remain a mystery. It has been shown that most of the islet-associated CD8 + CTLs in NOD mice are cytotoxic to beta cells in the context of the MHC class I molecule H-2K d , and that many of these CTLs express highly homologous T-cell receptor (TCR) a-subunit chains (Va17 and Ja42 elements joined by the N-region sequence M-R-D/E) 7,9,10 . By screening combinatorial peptide libraries, we have identi®ed two peptide ligands for this group of CTLs: NRP (amino-acid sequence KYNKANWFL), and NRP-A7 (KYNKANAFL), an alanine mutant analogue of NRP with superior agonistic properties 2 . As many of the CTL clones derived from islets of diabetic NOD mice recognize these peptides 2 , and as transgenic NOD mice expressing an NRP/ NRP-A7-reactive TCR (8.3-TCRab-transgenic NOD mice) become diabetic shortly after developing insulitis 8 , we thought that progres- sion of insulitis to diabetes might be driven by the accumulation of NRP/NRP-A7-reactive CTLs in islets. To follow the accumulation of antigen-speci®c CD8 + T cells in pancreatic islets of pre-diabetic NOD mice, we generated H-2K d tetramers containing the peptides NRP, NRP-A7, TUM (KYQAVTTTL, a negative control 11 ) or INS (LYLVCGERG, an insulin-derived peptide recognized by islet-associated T cells from young NOD mice 12 ). The NRP and NRP-A7 tetramers stained virtually all the splenic CD8 + T cells from 8.3-TCRab-transgenic NOD mice, but less than 1% of the splenic CD8 + T cells from NOD mice. The INS tetramer stained the INS-reactive CTL clone G9C8 (ref. 12), but neither this tetramer nor the negative control tetramer (TUM) stained the splenic CD8 + T cells from NOD or 8.3-TCRab- transgenic NOD mice (data not shown). As tetramer-reactive cells represent a very small fraction of all the T cells contained in freshly isolated islets (see Fig. 1 legend), we assayed for tetramer-reactive clonotypes within the cell population that has undergone antigen-driven activation and thus is likely to contain autoreactive speci®cities. Islets from non-diabetic 5-, 9-, 15- and 20-week-old NOD mice were cultured in the presence of recombinant interleukin-2 (rIL-2) for 6±7 days, to selectively expand IL-2 receptor-positive T cells, which constitute about 10% of all the islet-associated CD8 + T cells 7 . There were age-dependent increases in the absolute number of T cells recovered from NOD a TUM INS NRP NRP-A7 % Tetramer + CD8 + T cells Tetramer + CD8 + T cells (× 10 5 ) 5 9 15 20 0 5 10 15 20 25 1 1 c 0 0.2 0.4 0.6 5 9 15 20 b Tetramer-PE TUM NRP NRP-A7 CD8-FITC 8.3-TCR- transgenic NOD NOD 0.7 89 95 0.8 23 25 0.6 6 25 2 Age (weeks) Figure 1 TUM-, INS-, NRP- and NRP-A7-reactive CD8 + T cells derived from pancreatic islets. a, Percentage of tetramer-reactive T cells. Data (mean 6 s.e.m.) are from mice aged 5 weeks (n= 3), 9 weeks (n= 13), 15 weeks (n= 7) and 20 weeks (n= 11). P values: INS and NRP versus TUM at 5 weeks, P , 0.04; NRP and NRP-A7 versus TUM or INS at 9 weeks, P , 0.0006; NRP or NRP-A7 versus TUM or INS at 15 weeks, P , 0.013 and P , 0.003; NRP and NRP-A7 versus TUM at 20 weeks, P , 0.0001. `1', NRP-A7 versus NRP at 15 and 20 weeks, P , 0.05 and P , 0.008, respectively. Percentage of tetramer-reactive CD8 + cells in freshly isolated islets are 0.13 6 0.38% (n = 6; 5 weeks), 0.95 6 0.67% (n = 4; 9 weeks) and 1.6 6 0.5% (n = 2; 15 weeks) (NRP-A7); and 0.71 6 0.4 (n = 4; 5 weeks) and 0.31 6 0.4 (n = 4; 9 weeks) (INS). b, Absolute number (mean 6 s.e.m.) of tetramer-reactive T cells from the same samples as in a. P values: NRP-A7 at 15 and 20 weeks versus 9 and 5 weeks, P , 0.04; NRP and NRP-A7 versus TUM at 9 weeks, P , 0.001; NRP or NRP-A7 versus INS at 9 weeks, P , 0.035; NRP or NRP-A7 versus TUM or INS at 15 weeks, P , 0.037; NRP or NRP-A7 versus TUM at 20 weeks, P , 0.0001. `2', NRP-A7 versus NRP at 20 weeks, P , 0.05. c, Representative tetramer staining patterns. Over 90% of the CD8-negative cells shown in the plots are CD4 + . © 2000 Macmillan Magazines Ltd