Restricted islet-cell reactive T cell repertoire of early pancreatic islet infiltrates in NOD mice - PubMed (original) (raw)

Restricted islet-cell reactive T cell repertoire of early pancreatic islet infiltrates in NOD mice

Felix J Baker et al. Proc Natl Acad Sci U S A. 2002.

Abstract

The mechanisms responsible for initiating autoimmune diabetes remain obscure. Here, we describe a method for identifying both the alpha- and beta-chains of the T cell receptor (TCR) from individual pancreatic islet-infiltrating T cells at the earliest stages of disease in nonobese diabetic mice (NOD). Analysis of the TCR repertoire of these early islet infiltrates reveals enrichment for a small subset of TCR sequences. Reconstitution of these TCR in vitro demonstrates that these receptors confer reactivity to islet cells but not to the well characterized autoantigens, glutamic acid decarboxylase (GAD65) and insulin. Thus, autoimmune diabetes in NOD may be initiated by a limited number of antigens distinct from GAD65 and insulin.

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Figures

Figure 1

Figure 1

Single-cell PCR method for TCR amplification. (A) Nested PCR strategy for specific amplification of TCR α- and β-chains from single cells. The thin lines connecting V(D)J segments represent areas of N-region addition. Regions are not drawn to scale. The upper set of arrows indicates the relative position of external oligonucleotides used in the first round of PCR. The lower set of arrows represents the nested position of the internal oligonucleotides used in the second round of PCR. (B) Sample PCR amplification of TCR α- and β-chains from 12 single CD4+ T cells using internal and external oligonucleotide pools. Completed second-round reactions were electrophoresed through a 1.5% agarose gel and stained with ethidium bromide. MW, molecular weight markers (100-bp ladder).

Figure 2

Figure 2

CDR3 sequences for Vβ8.2 TCR from early islet infiltrates in NOD. Amino acid sequence is presented with junctionally encoded sequences indicated in bold face. Vβ, Jβ usage, and CDR3 length are given in the three columns at the left of the figure. The presence of an aspartate or glutamate at position 2 of the CDR3 is highlighted.

Figure 3

Figure 3

Vβ1 motif in early islet infiltrates in NOD. (A) CDR3 sequences for Vβ1 TCR from early islet infiltrates (Islets). Sequences from T cells in pancreas-draining lymph nodes (LN) from the same animals are included (Lower) for comparison. The presence of glutamine in position 2 of the CDR3 is indicated in light gray. Aspartate or glutamate at position 2 or 3 of the CDR3 is highlighted in dark gray. (B) Paired α-chain CDR3 sequences for 5 Vβ1 sequences. The presence of a junctionally encoded arginine at position 1 or 2 of the CDR3 is highlighted. (C) Vβ1 sequences from the islet infiltrates of 11-week-old NOD. (A_–_C) Amino acid sequence is presented with junctionally encoded sequences indicated in bold face. Vβ usage, Jβ usage, and CDR3 length are given in the three columns at the left of each panel. (A and_B_) *, denotes the sequence used for reconstitution experiments in Fig. 6.

Figure 4

Figure 4

Vβ12 motif in early islet infiltrates in NOD. (A) CDR3 sequences for Vβ12 TCR from early islet infiltrates (Islets). Sequences from T cells in pancreas-draining lymph nodes (LN) from the same animals are included (Lower) for comparison. The presence of serine in position 1 of the CDR3 is indicated in light gray. Leucine at position 2 of the CDR3 is highlighted in dark gray. (B) Paired α-chain CDR3 sequences for 3 Vβ12 sequences. (C) Vβ12 sequences from the islet infiltrates of 11-week-old NOD. (A_–_C) Amino acid sequence is presented with junctionally encoded sequences indicated in bold face. Vβ usage, Jβ usage, and CDR3 length are given in the three columns at the left of each panel. (_A_and B) *, denotes the sequence used for reconstitution experiments in Fig. 6.

Figure 5

Figure 5

TCR β-chain variable region usage in (A) islet-infiltrating CD4+ T cells from 14- to 18-day-old NOD mice and in (B) peripheral CD4+ T cells from the same animals. (A) Full-length sequences (74) which could be clearly classified were obtained by the single-cell PCR protocol described in this report. (B) The relative frequency of β-chain variable regions was established by FACS staining. ND, not determined because antibodies are not currently available.

Figure 6

Figure 6

In vitro reconstitution of Vβ1Vα4 and Vβ12Vα1. (A) Reactivity to islet cells presented by I-Ag7 splenocytes. (B) Reactivity to MCC (88–103) peptide presented by I-Ek splenocytes. (C) Reactivity to GAD65 and insulin β-chain presented by I-Ag7 splenocytes. The GAD65 protein used in_C_ stimulates T cell hybridomas specific for GAD65 in the context of I-Ag7 in parallel experiments (M.L. and M.M.D., unpublished data). The recombinant insulin used in C is a commercially available reagent (Sigma). (A_–_C) Stimulation index corresponds to T cell proliferation in the presence of APC and the indicated amount of antigen normalized to the degree of proliferation observed in the presence of APC alone. The experiments for_A_–C were conducted in parallel by using the same APC and T cells. Data shown is the average value for at least two samples, with data values generally varying by less than 20%.

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