To determine if endogenous TCR expression enhanced positive selection of CD4+ KRN T cells about I-Ab, we bred TCR/deficient KRN.H-2b mice. no apparent gene-dosage effect (data not demonstrated). The TCR/-deficient KRN.H-2b/g7 mice had a significant reduction in the frequency and complete quantity of CD4 SP thymocytes and CD4+ splenic T cells relative to mice able to express endogenous TCR and/or TCR chains, as well as a reduction in the level of arthritogenic PR52B anti-GPI IgG autoantibodies (Figure 3 and Supplemental Figure 1 (S1)). Open in a separate window Number 2 Both endogenous TCR and TCR chains can facilitate spontaneous autoimmune arthritis(A) Arthritis development expressed as ankle thickening over time in mice of ELQ-300 the indicated genotypes (all KRN.H-2b/g7). The + denotes the endogenous and alleles are present either in heterozygosity or homozygosity; ? denotes homozygosity of the knockout allele. Each collection represents one mouse (= 5C6 mice per group). (B) Representative ankle sections from 8-week-old mice of the indicated genotypes. H&E staining; level bar in remaining panel represents 500 microns and all images are at the same magnification. Open in a separate window Number 3 Endogenous TCR/ manifestation allows autoreactive CD4+ T cells to escape clonal deletionFlow cytometry of thymocytes (top) and CD3+ splenocytes (bottom) from mice of the indicated genotypes (all KRN.H-2b/g7) and stained with anti-CD4 and anti-CD8 monoclonal antibodies. Figures show the percentage of cells in each of the boxed gates. The plots are representative of 6 experiments with a total of 5C6 mice/group. The mice were 8C10 weeks of age. Because the development of arthritis with this model depends on autoantibody production, we wanted to verify that the lack of arthritis development in the TCR/-deficient KRN.H-2b/g7 mice was not due to an unanticipated B cell defect. Adoptive transfer of CD4+ splenic T cells from KRN.H-2b mice to the TCR/-deficient KRN.H-2b/g7 mice provoked the quick onset of arthritis (Supplemental Table I), demonstrating the failure of the TCR/-deficient KRN.H-2b/g7 mice to develop arthritis was because of the lack of CD4+ KRN T cells. Taken together, these findings show that incomplete TCR (or TCR) allelic exclusion facilitates the thymic maturation of CD4+ T cells expressing the autoreactive KRN TCR, allowing ELQ-300 them to provoke autoantibody-mediated arthritis. Dual TCR manifestation is readily recognized among CD4 SP thymocytes To determine how dual TCR manifestation enhances thymic maturation of autoreactive KRN T cells, we performed further analyses to determine at what stage of T cell differentiation dual TCR manifestation was first detectable. Manifestation of endogenously-encoded TCR and TCR chains was readily recognized among CD4+ splenic T cells (Supplemental Number 2) and CD4 SP thymocytes (Supplemental Number 3). Among splenocytes, the rate of recurrence of cells expressing dual TCR chains was higher in mice lacking endogenous TCR chains relative to the endogenous TCR/-adequate mice ELQ-300 (34.7% versus 18.3%); similarly the rate of recurrence of cells expressing dual TCR chains was higher in mice lacking endogenous TCR chains relative to the endogenous TCR/-adequate mice (41.7% versus 32.4%), further demonstrating that allelic inclusion can occur readily in the KRN system. Cells co-expressing an endogenously-encoded TCR chain plus an endogenously-encoded TCR chain in addition to the KRN-encoded TCR chain were detectable, but not common. Manifestation of dual TCR chains was associated with decreased manifestation of the KRN transgene-encoded V6 ELQ-300 chain in the presence of I-Ag7 (Numbers 1, S2) (23). ELQ-300 We did not detect significant endogenous TCR or TCR manifestation at the earlier CD4+ CD8+ double-positive (DP) and CD4? CD8? double-negative (DN) phases of thymic development (Number S4 and data not shown). The simplest interpretation of these findings is definitely that dual TCR manifestation is rare through the DP stage, but that dual TCR-expressing DP thymocytes.