Non-obese diabetic mice select a low-diversity repertoire of natural regulatory T cells

Abstract

Thymus-derived Foxp3(+) natural regulatory CD4 T cells (nTregs) prevent autoimmunity through control of pathogenic, autoreactive T cells and other immune effector cells. Using T cell receptor (TCR) transgenic models, diversity within this lineage has been found to be similar to that of conventional CD4 T cells. To determine whether balanced TCR diversity may be perturbed in autoimmunity, we have analyzed receptor composition in C57BL/6 and autoimmune non-obese diabetic (NOD) mice. The natural regulatory and conventional CD4 repertoires of C57BL/6 had similar diversities. Despite the apparently normal thymic development of the NOD nTreg lineage, TCR diversity within the selected repertoire was markedly restricted. Detailed analysis of TCRalpha and -beta chain composition is consistent with positive selection into the natural regulatory lineage being under stringent audition for interaction with MHC class II/self-peptide. The NOD MHC region, including the unique H2-A(g7) class II molecule, partly accounts for the reduction in diversity, but additional NOD genetic contribution(s) are required for complete repertoire compaction. Mechanistic links between MHC, autoimmunity, and nTreg diversity identified in this study are discussed.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Low-diversity AV9 TCR repertoire in NOD thymic nTregs. Thymic nTregs (CD25+CD4+) and conventional SP4 (CD25−CD4+) thymocytes from C57BL/6 (A and B) and NOD (C and D) mice were sorted and the frequency of individual AV9 TCR rearrangements determined. Each unique CDR3 sequence is represented by a segment proportional to its frequency. Repertoire diversity (D value) is shown for each population. Color is used for clarity only and does not represent a particular sequence. Number of sequences collected for each population is shown in Table S1. Data are derived from a single representative experiment of n = 2 (C57BL/6) and n = 3 (NOD) independent experiments.

Fig. 2.

J segment usage is highly restricted among AV9 NOD thymic nTregs. J segment usage within thymic nTreg (CD4+CD25+) and Tconv (CD4+CD25−) thymocytes from C57BL/6 (A and B) and NOD (C and D) mice. The percentage of the AV9 repertoire using each J segment is shown by bar height. J segments are represented on the x axis following the genomic order. Type I and type II J segments are shown as open and closed bars, respectively. Percentages of rearrangements occurring outside the recombination blueprint are indicated. Number of sequences collected for each population is shown in Table S1. Data are derived from a single representative experiment of n = 2 (C57BL/6) and n = 3 (NOD) independent experiments.

Fig. 3.

Low diversity and restriction of J segment use in AV12 TCR repertoire of NOD thymic nTregs. Thymic nTreg (CD4+CD25+) and Tconv (CD25−CD4+) thymocytes from C57BL/6 (A and B) and NOD (C and D) mice were sorted and the frequency of individual AV12 TCR sequences determined. The frequency of each unique CDR3 sequence correlates with segment size; color is used for clarity only and does not represent a particular sequence. Repertoire diversity (D value) is shown for each population.

Fig. 4.

Restricted J segment usage amongst AV12 NOD thymic nTregs. J segment usage within thymic nTreg (CD4+CD25+) and Tconv (CD4+CD25−) thymocytes from C57BL/6 (A and B) and NOD (C and D) mice. The percentage of the AV12 repertoire using each J segment is shown by bar height. J segments are represented on the x axis following the genomic order. Type I and type II J segments are shown as open and closed bars, respectively.

Fig. 5.

Diversity of BV8.2-BJ2.3 TCR repertoire in NOD thymic nTregs. Thymic nTreg (CD4+CD25+) and Tconv (CD4+CD25−) thymocytes from C57BL/6 (A and B) and NOD (C and D) mice were sorted and the frequency of individual BV8.2-BJ2.3 TCR sequences determined. Each unique CDR3 sequence is represented by a segment proportional to its frequency; color is used for clarity only and does not represent a particular sequence. Repertoire diversity (D value) is shown for each population. Number of sequences collected for each population is shown in Table S3.

Fig. 6.

C57BL/6g7 mice have a nTreg repertoire distinct from NOD. nTreg (CD4+CD25+) and Tconv (CD4+CD25−) thymocytes from B6g7 were sorted (A and B) and the frequency of individual AV9 TCR sequences determined. The frequency of each unique CDR3 sequence correlates with segment size. Color is used for clarity only and does not represent a particular sequence. Corresponding J segment use is represented in the lower panels (C and D). The proportion of J segments outside the region of preferential recombination and relative use of type I (open bars) and type II (closed bars) J segments is shown.