Incomplete TCR-β allelic exclusion accelerates spontaneous autoimmune arthritis in K/BxN TCR transgenic mice

Abstract

Allelic exclusion of antigen receptor loci is a fundamental mechanism of immunological self-tolerance. Incomplete allelic exclusion leads to dual T-cell receptor (TCR) expression and can allow developing autoreactive αβ T lymphocytes to escape clonal deletion. Because allelic exclusion at the TCR-β locus is more stringent than at the TCR-α locus, dual TCR-β expression has not been considered a likely contributor to autoimmunity. We show here that incomplete TCR-β allelic exclusion permits developing thymocytes bearing the autoreactive, transgene-encoded KRN TCR to be positively selected more efficiently, thereby accelerating the onset of spontaneous autoimmune arthritis. Our findings highlight dual TCR-β expression as a mechanism that can enhance the maturation of autoreactive pathogenic T cells and lead to more rapid development of autoimmune disease.

Figure 1. Dual TCRβ expression is common in KRN TCR transgenic mice

Flow cytometric analysis of CD4 SP thymocytes and CD4⁺ splenic T cells of the indicated mice is shown. The KRN transgene-encoded Vβ6 chain is on the x-axis. Staining with a panel of Vβ antibodies is represented on the y-axis (Vβ2, 3, 4, 5.1/5.2, 7, 8.1/8.2, 8.3, 9, 10b, 11, 12, 13, 14, 17a). Numbers indicate percentages; cells expressing dual TCRβ chains are represented in the top right quadrant of each plot (gray font). The results are representative of data from two independent experiments.

Figure 2. Both endogenous TCRα and TCRβ chains can facilitate spontaneous autoimmune arthritis

(A) Arthritis development expressed as ankle thickening over time in mice of the indicated genotypes (all KRN.H-2^b/g7). The “+” denotes that the endogenous Tcra and Tcrb alleles are present either in heterozygosity or homozygosity; “−“ denotes homozygosity of the knockout allele. Each line represents one mouse (n = 5–6 mice per group). (B) Representative ankle sections from 8-week-old mice of the indicated genotypes. H&E staining; scale bar in left panel represents 500 microns and all images are at the same magnification.

Figure 3. Endogenous TCRα/β expression allows autoreactive CD4⁺ T cells to escape clonal deletion

Flow cytometry of thymocytes (top) and CD3⁺ splenocytes (bottom) obtained from mice of the indicated genotypes (all KRN.H-2^b/g7) and stained with anti-CD4 and anti-CD8 monoclonal antibodies. Numbers indicate the percentage of cells in each of the boxed gates. The plots are representative of 6 experiments with a total of 5–6 mice/group. The mice were 8–10 weeks of age.

Figure 4. Endogenous TCRα/β expression promotes positive selection of KRN CD4⁺ T cells

Flow cytometry of thymocytes (top) and CD3⁺ splenocytes (bottom) from mice of the indicated genotypes (all KRN.H-2^b) and stained with anti-CD4 and anti-CD8 monoclonal antibodies. Numbers indicate the percentage of cells in each of the boxed gates. Mice were 8–16 weeks of age; within this range, age-related differences were not apparent except in the TCRα/TCRβ-deficient mice, as shown. Gates were set based on isotype control staining within individual experiments (e.g. the panels in the 4^th column are from a separate experiment from the others). The plots are representative of 3 experiments with a total of 4–6 mice/group.

Figure 5. KRN.H-2^b/g7 mice lacking endogenous TCRα and TCRβ chains develop late-onset arthritis as autoreactive T cells escape clonal deletion

(A) Arthritis development expressed as ankle thickening over age in Tcra⁻ Tcrb⁻ KRN.H-2^b/g7 mice. Each line represents one mouse. (B) Representative ankle section from an arthritic Tcra⁻ Tcrb⁻ KRN.H-2^b/g7 mouse. H&E staining; scale bar represents 500 microns. (C) Flow cytometry of thymocytes and CD3⁺ splenocytes from an arthritic 25-week-old Tcra⁻ Tcrb⁻ KRN.H-2^b/g7 mouse, stained with anti-CD4, anti-Vβ6 (the KRN clonotype), and a mixture of antibodies recognizing 3 endogenous Vα chains and 3 endogenous Vβ chains. Numbers indicate the percentage of cells in each box. The plots on the right depict the cells in the red box. Gating was determined based on control mice with cell populations present in each of the gates.

Figure 6. Endogenous TCRα/β expression is required for autoimmune endocarditis in KRN.H-2^b/g7 mice

(A) Representative mitral valves of mice of the indicated genotypes. H&E, scale bar represents 100 microns. (B) The maximum mitral thickness of the mitral valve is depicted. Circles represent individual mice, bars represent the mean; P-values were calculated using Student’s t-test.