The frequency of circulating CD3+ cells was 23.0 ± 6.4% of peripheral blood mononuclear cells (PBMC) for the control group and 27.9 ± 10.8% for the Aire group; the difference was not statistically significant. This indicated significant reconstitution of the T cell compartment

in both recipient groups, as the normal frequency of CD3+ selleck chemicals llc T cells in the blood of WT C57BL/6 mice has been reported to be 21% [35]. At the time of termination, we determined the clinical status of the recipients using a clinical score adapted from Cooper et al. [34]. There was no difference between the groups in weight loss or hunching, symptoms that generally indicate wasting. Overall, the clinical scores were similarly low in both groups Dasatinib manufacturer (data not shown) and in both the Aire and control groups the animals remained clinically healthy. Only two animals in the control group lost over 10%

of their original body weight. The recipients were euthanized 2 months after the transfer and tissues harvested for detailed analysis. In PBMC, the frequency of CD3+ cells was comparable in the Aire and control group (25.6 ± 12.0% and 21.9 ± 21.5%, respectively), but the frequency of CD3+ cells expressing the cell cycle marker Ki-67 was significantly higher in the Aire group (Fig. 1A). Similarly, in spleen the frequency of Ki-67+ cells within the CD3+ population was higher in the Aire group (Fig. 1B), and this also resulted in the accumulation of CD3+ splenocytes at a higher frequency (Fig. 1C). In both blood and spleen of the Aire group recipients, the increased expression of Ki-67 was found particularly in the CD8+ T cells (Fig. 1D,E), and in the spleen the Aire group had a significantly higher frequency of CD8+ cells within the CD3+ population (Fig. 1F). Together, these

data show that cells originating from Aire−/− donors hyperproliferate in response to lymphopenia, and that CD8+ T cells are mostly responsible for this hyperproliferation. At the time of termination, we collected tissues reported GBA3 to be targets of the autoimmune attack in Aire−/− animals [9, 10, 12]. Histological analysis of stomach, adrenals, ovaries, liver, salivary glands and pancreas showed low-level lymphocytic infiltrates in the three last-mentioned tissues, but no significant differences were observed between the recipient groups. To confirm this lack of difference with a more quantitative method, we used qPCR to measure the amount of T cell receptor gene constant alpha (TCR Cα) mRNA, normalized against the house-keeping gene Hprt mRNA levels. Again, we did not see any significant difference between the recipient groups (Fig. 2), confirming that T cells were present at equal numbers in the tissues studied in both groups. It has been reported that liver infiltrates in Aire−/− mice consist mainly of B cells [26], so we also measured the amount of CD19 mRNA in the liver tissue of the recipients.