Restricted use of T cell receptor (TCR) gene segments is characteristic of several induced autoimmune disease models. TCR sequences have previously been unavailable for pathogenic T cells which react with a defined autoantigen in a spontaneous autoimmune disease. The majority of T cell clones, derived from islets of NOD mice which spontaneously develop type I diabetes, react with insulin peptide B-(9-23). We have sequenced the alpha and beta chains of TCRs from these B-(9-23)-reactive T cell clones. No TCR beta chain restriction was found. In contrast, the clones (10 of 13) used V alpha13 coupled with one of two homologous J alpha segments (J alpha45 or J alpha34 in 8 of 13 clones). Furthermore, 9 of 10 of the V alpha13 segments are a novel NOD sequence that we have tentatively termed V alpha13.3. This dramatic alpha chain restriction, similar to the beta chain restriction of other autoimmune models, provides a target for diagnostics and immunomodulatory therapy.

Annals of the New York Academy of SciencesVolume 778, Issue 1 p. 371-372 Intranasal Administration of Insulin Peptide B: 9–23 Protects NOD Mice from Diabetes DYLAN DANIEL, DYLAN DANIEL Barbara Davis Center for Childhood Diabetes and Interdepartmental Program of Immunology Biophysics, and Genetics University of Colorado Health Sciences Center 4200 East 9th Avenue Box B-140 Denver, Colorado 80220Search for more papers by this authorDALE R. WEGMANN, DALE R. WEGMANN Barbara Davis Center for Childhood Diabetes and Department of Immunology Biophysics, and Genetics University of Colorado Health Sciences Center 4200 East 9th Avenue Box B-140 Denver, Colorado 80220 Department of Biochemistry, Biophysics, and Genetics University of Colorado Health Sciences Center 4200 East 9th Avenue Box B-140 Denver, Colorado 80220Search for more papers by this author DYLAN DANIEL, DYLAN DANIEL Barbara Davis Center for Childhood Diabetes and Interdepartmental Program of Immunology Biophysics, and Genetics University of Colorado Health Sciences Center 4200 East 9th Avenue Box B-140 Denver, Colorado 80220Search for more papers by this authorDALE R. WEGMANN, DALE R. WEGMANN Barbara Davis Center for Childhood Diabetes and Department of Immunology Biophysics, and Genetics University of Colorado Health Sciences Center 4200 East 9th Avenue Box B-140 Denver, Colorado 80220 Department of Biochemistry, Biophysics, and Genetics University of Colorado Health Sciences Center 4200 East 9th Avenue Box B-140 Denver, Colorado 80220Search for more papers by this author First published: February 1996 https://doi.org/10.1111/j.1749-6632.1996.tb21146.xCitations: 31AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat No abstract is available for this article. REFERENCES 1 Makino, S., K. Kunimoto, Y. Muraoka, Y. Mizushima, K. Katagiri & Y. Tochino. 1980. Exp. Anim. 29: 1–13. 2 Wegmann, D. R., M. Norbury-Glaser & D. Daniel. 1994. Eur. J. Immunol. 24: 1853–1857. 3 Daniel, D., R. G. Gill, N. Schloot & D. Wegmann. 1995. Eur. J. Immunol. 25: 1056–1062. 4 Wegmann, D. R., R. G. Gill, M. Norbury-Glaser, N. Schloot & D. Daniel. 1994. Analysis of the spontaneous T cell response to insulin in NOD mice. J. Autoimmunity 7: 833–843. 5 Zhang, Z. J., L. Davidson, G. Eisenbarth & H. L. Weiner. 1991. Proc. Natl. Acad. Sci. USA 88: 10252–10256. 6 Atkinson, M. A., N. K. MacLaren & R. Luchetta. 1990. Diabetes 39: 933–937. 7 Mur, A., A. Peck, Clare-Salzler, Y.-H. Song, J. Corneliius, R. Luchetta, J. Krischer & N. MacLaren. 1995. J. Clin. Invest. 95: 628–634. 8 Hoyne, G. F., R. E. O'Hehir, D. C. Wraith, W. R. Thomas & J. R. Lamb. 1993. J. Exp. Med. 178: 1783–1788. Citing Literature Volume778, Issue1Oral Tolerance: Mechanisms and ApplicationsFebruary 1996Pages 371-372 ReferencesRelatedInformation

The observation that overt type I diabetes is often preceded by the appearance of insulin autoantibodies and the reports that prophylactic administration of insulin to biobreeding diabetes-prone (BB-DP) rats, nonobese diabetic (NOD) mice, and human subjects results in protection from diabetes suggest that an immune response to insulin is involved in the process of beta cell destruction. We have recently reported that islet-infiltrating cells isolated from NOD mice are enriched for insulin-specific T cells, that insulin-specific T cell clones are capable of adoptive transfer of diabetes, and that epitopes present on residues 9-23 of the B chain appear to be dominant in this spontaneous response. In the experiments described in this report, the epitope specificity of 312 independently isolated insulin-specific T cell clones was determined and B-(9-23) was found to be dominant, with 93% of the clones exhibiting specificity toward this peptide and the remainder to an epitope on residues 7-21 of the A chain. On the basis of these observations, the effect of either subcutaneous or intranasal administration of B-(9-23) on the incidence of diabetes in NOD mice was determined. The results presented here indicate that both subcutaneous and intranasal administration of B-(9-23) resulted in a marked delay in the onset and a decrease in the incidence of diabetes relative to mice given the control peptide, tetanus toxin-(830-843). This protective effect is associated with reduced T-cell proliferative response to B-(9-23) in B-(9-23)-treated mice.

The majority of T cell clones derived from islets of NOD mice react with insulin, and 97% of these clones react with an immunodominant peptide termed B2 (amino acids 9 to 23 of the insulin B chain). These clones are CD4 positive and restricted by I-Ag7 and are able to transfer diabetes into young NOD mice and destroy transplanted human islets. Administration of insulin, insulin B chain, insulin B2 peptide, or genetic introduction of insulin into NOD mice all prevent diabetes. With the potential importance of insulin reactivity we have begun the sequencing of the T cell receptors of anti-insulin T cell clones. To date, sequencing is complete on four clones derived from three different mice. Four different Vβ (2,6,12,14) and four Jβ elements with different NDN junctions have been found. Three different Vα chains were utilized (3, 10, and two 13). In contrast to the above heterogeneity, the first two sequenced clones utilized the same Jα (45) with a KLTFGKGT octamer. The next two clones contained Jα 9 and Jα 34, which are the only other two Jαs (out of a reported 49) sharing the KLTFGKGT octamer. As reported in the literature, this Jα octamer was utilized by 0/4 non-insulin reactive diabetogenic CD4 NOD clones and 1/5 islet reactive CD4 clones with unknown specificity. Thus far, we have also sequenced two islet-derived GAD reactive clones and both utilize Jα chains (6, 40) lacking the octamer. This data suggests that the Jα octamer KLTFGKGT may relate to B2 peptide specificity and islet targeting. Both the Jα octamer and the insulin B2 peptide are preserved in the human genome, suggesting that a similar process may occur in type I diabetes.

The non-obese diabetic (NOD) mouse develops diabetes as a result of spontaneous T cell mediated destruction of the insulin-producing beta-cells. Tolerization to glutamic acid decarboxylase (GAD65) has been reported to inhibit spontaneous T cell proliferative responses to GAD65 and GAD65 peptides and prevent insulitis and diabetes in NOD mice. To evaluate the role of T cells responsive to GAD65 in induction of diabetes in NOD mice we generated T cell clones from spleen cells of three prediabetic NOD mice using the reported immunodominant human GAD65 peptides nos. 17, 34 and 35, which are spontaneously recognized by NOD spleen cells. The ten T cell clones established from two female and one male NOD mice recognized either the GAD65 peptide no. 35 which has an identical amino acid sequence in mice and humans or recognized the human GAD65 peptide no. 17 which is different in two amino acids from murine GAD65 peptide no. 17. None of the clones exhibited responses to islet cells, and GAD65 peptide no. 17 responsive clones did not cross react with the murine GAD65 peptide no. 17. All clones were CD4 positive and expressed the α/β T cell receptor, but differed in their Vβ usage. Analysis ofin vitroproduction of IFNγ, IL-2 and IL-4 demonstrated a TH1 and TH0 like functional subset of the individual clones.In vivo, neither the autoreactive T cell clones specific for GAD65 peptide no. 35 nor the xenoreactive clones specific for GAD65 peptide no. 17 were able to accelerate diabetes in young NOD mice or transfer diabetes into NODscidmice.

Insulin-specific T cells have been found to be present in high frequency among nominally islet-cell-specific T cells in the islet infiltrates that accumulate in NOD mice. In a previous report in which clones obtained from 7- and 12-week-old mice were examined, we identified a 15-residue peptide of the B chain as the dominent epitope for this response. Despite the fact that the response to insulin appears to be directed toward this single peptide, diverse TCR V beta usage was observed. That insulin-specific T cells contribute to beta cell damage is suggested by the fact that all clones tested could mediate beta cell destruction upon adoptive transfer. In the present report we extend this examination of insulin-specific T cells to lines and clones established from mice ranging in age from 4-12 weeks. These clones were found to be very similar to those from 7- and 12-week-old mice. The response was directed to the same peptide and most were found to produce IFN gamma, but none produced IL-4.

One impediment to detailed characterization of islet-specific T cells from the NOD mouse model of diabetes is the difficulty encountered in isolation of such cells. This report describes a method that allows routine isolation of relatively large numbers of T cells highly enriched for reactivity toward islet antigens. The method involves renal subcapsular transplantation of spontaneously diabetic NOD mice with NOD islets. These grafts are rapidly destroyed in a tissue-specific manner and this destruction is accompanied by lymphocytic infiltration. Here we demonstrate that the islet graft infiltrates are an excellent source of islet-specific T cells and that islet-specific T-cell lines and clones can be established from these cells. Islet-specific T-cell clones isolated from a T-cell line established from the islet graft infiltrates were capable of adoptive transfer of diabetes to NOD/LtSz-scid recipients.