T-cell regulation by casitas B-lineage lymphoma (Cblb) is a critical failsafe against autoimmune disease due to autoimmune regulator (Aire) deficiency

Abstract

Autoimmune polyendocrinopathy syndrome type 1 (APS1) results from homozygous Aire mutations that cripple thymic deletion of organ-specific T cells. The clinical course in man and mouse is characterized by high variability both in the latent period before onset of autoimmune disease and in the specific organs affected, but the reasons for this are unknown. Here we test the hypothesis that the latent period reflects the failsafe action of discrete postthymic mechanisms for imposing self-tolerance in peripheral T cells. Aire-deficient mice were crossed with mice of a uniform major histocompatibility complex (MHC) haplotype and genetic background carrying specific genetic defects in one of four distinct peripheral tolerance mechanisms: activation-induced cell death (Fasl(gld/gld)), anergy and requirement for CD28 costimulation (Cblb(-/-)), inhibition of ICOS and T(FH) cells (Rc3h1(san/san)), or decreased numbers of Foxp3(+) T regulatory cells (Card11(unm/unm)). Cblb-deficiency was unique among these four in precipitating rapid clinical autoimmune disease when combined with Aire-deficiency, resulting in autoimmune exocrine pancreatitis with median age of survival of only 25 d. Massive lymphocytic infiltration selectively destroyed most of the exocrine acinar cells of the pancreas and submandibular salivary gland, and CD4(+) and CD8(+) subsets were necessary and sufficient to transfer the disease. Intrinsic regulation of peripheral T cells by CBL-B thus serves a uniquely critical role as a failsafe against clinical onset of autoimmune disease in AIRE deficiency, and multiple peripheral tolerance mechanisms may need to fail before onset of clinical autoimmunity to many organs.

Fig. 1.

Effects of combined defects in central and peripheral tolerance mechanisms. Survival measured from birth to 20 wk for mice with single and double mutations in: (A) Aire and Card11; (B) Aire and Fasl; (C) Aire and Rc3h1; (D) Aire and Cblb. All mice were on the B10.BR (H2^k) genetic background (n ≥ 6 for each group). (E) Body weight at 2, 3, 4, and 5 wk after birth of male and female mice of the following genotypes: Aire^+/+Cblb^+/+ (n = 4 males, 4 females); Aire^+/+Cblb^−/− (n = 7 males, 7 females) and Aire^−/−Cblb^+/+ (n = 4 males, 4 females) or Aire^−/−Cblb^−/− (n = 7 males, 7 females). Statistical analysis by One-way ANOVA comparing all groups, followed by pairwise Bonferroni post-test. *, **, *** denote groups where the probability of no difference was P < 0.05, P < 0.01, or P < 0.001, respectively. Error bars represent s.d. (F and G) Runted appearance and pancreatic atrophy of a representative 24-d female Aire^−/−Cblb^−/− mouse (F, Upper; G, Left) compared with a littermate control (F, Lower; G, Right). Sto, stomach; Sp, spleen; Kd, kidney; Panc, pancreas.

Fig. 2.

Lymphocytic exocrine pancreatitis and sialoadenitis in Aire^−/−Cblb^−/− mice. (A) Representative H&E stained sections of pancreas (Upper) and submandibular salivary gland (Lower) from 3- to 6-wk-old Aire^−/−Cblb^−/− mice (taken at time of death or euthanasia) and 20-wk-old control counterparts (endpoint of study) of the indicated genotypes. (Scale bars: 200 μm.) (n = 10 for each group). (B) Higher power images of pancreas from Aire^−/−Cblb^−/− mice. (Left) Grade 5 pancreatitis; (Right) grade 3 pancreatitis. (Scale bars: 200 μm.) (C) Submandibular salivary gland from Aire^−/−Cblb^−/− mice. (Left) Grade 5 sialoadenitis; (Right) grade 1 sialoadenitis. (Scale bars: 200 μm.) (D) Percentage of mice of the indicated genotypes with different grades of pancreatitis, sialoadenitis, and gastritis (n = 10 for each group, all age 20 wk except Aire^−/−Cblb^−/− mice 3–6 wk old). Pathology was scored blinded according to the scale shown in Fig. S1.

Fig. 3.

Transfer of pancreatitis by T lymphocytes from Aire^−/−Cblb^−/− mice. (A and B) Flow cytometric analysis of splenocytes from littermate 29 d mice of the indicated genotypes. Data are representative of three independent experiments analyzing whole litters. (C) Survival of Rag1-deficient B10.BR mice after adoptive transfer of 1–9 × 10⁶ splenocytes from Aire^−/−Cblb^−/− or wild-type mice. (D) Percentage of recipient mice in (C) with different grades of pancreatitis. (E) Percentage of CD4⁺ or CD8⁺ T cells in blood lymphocytes of mice in (C) 12 d posttransfer. Data in (C–E) are representative of two independent experiments (n ≥ 4 for each group). (F) Survival of Rag1-deficient B10.BR mice after adoptive transfer of 2 × 10⁶ unfractionated spleen cells (containing 2 × 10⁵ CD4 or 1 × 10⁵ CD8 cells), 2 × 10⁶ spleen cells selectively depleted of CD4- or CD8-cells, or 2 × 10⁵ CD4- or 1 × 10⁵ CD8-enriched spleen cells, obtained from bone marrow chimeras of the indicated genotypes. Recipients of CD4-depleted or CD8-enriched cells, and recipients of CD8-depleted or CD4-enriched cells, were also treated with depleting antibody to CD4 or CD8, respectively, on days 0, 1, 7, and 14 to deplete any residual T cells of the relevant subset. (G) Percentage of recipient mice in F with different grades of exocrine pancreatitis.