Inducible de novo expression of neoantigens in tumor cells and mice.

Inducible expression of neoantigens in mice would enable the study of endogenous antigen-specific naïve T cell responses in disease and infection, but has been difficult to generate because leaky antigen expression in the thymus results in central T cell tolerance. Here we develop inversion-induced joined neoantigen (NINJA), using RNA splicing, DNA recombination and three levels of regulation to prevent leakiness and allow tight control over neoantigen expression. We apply NINJA to create tumor cell lines with inducible neoantigen expression, which could be used to study antitumor immunity. We also show that the genetic regulation in NINJA mice bypasses central and peripheral tolerance mechanisms and allows for robust endogenous CD8 and CD4 T cell responses on neoantigen induction in peripheral tissues. NINJA will enable studies of how T cells respond to defined neoantigens in the context of peripheral tolerance, transplantation, autoimmune diseases and cancer.

Notch3 is dispensable for thymocyte ß-selection and Notch1-induced T cell leukemogenesis.

Notch1 (N1) signaling induced by intrathymic Delta-like (DL) ligands is required for T cell lineage commitment as well as self-renewal during "ß-selection" of TCRß⁺CD4⁻CD8⁻ double negative 3 (DN3) T cell progenitors. However, over-expression of the N1 intracellular domain (ICN1) renders N1 activation ligand-independent and drives leukemic transformation during ß-selection. DN3 progenitors also express Notch3 (N3) mRNA, and over-expression of ligand-independent mutant N3 (ICN3) influences ß-selection and drives T cell leukemogenesis. However, the importance of ligand-activated N3 in promoting ß-selection and ICN1-induced T cell leukemogenesis has not been examined. To address these questions we generated mice lacking functional N3. We confirmed that DN3 progenitors express N3 protein using a N3-specific antibody. Surprisingly however, N3-deficient DN3 thymocytes were not defective in generating DP thymocytes under steady state conditions or in more stringent competition assays. To determine if N3 co-operates with N1 to regulate ß-selection, we generated N1;N3 compound mutants. However, N3 deficiency did not exacerbate the competitive defect of N1⁺/⁻ DN3 progenitors, demonstrating that N3 does not compensate for limiting N1 during T cell development. Finally, N3 deficiency did not attenuate T cell leukemogenesis induced by conditional expression of ICN1 in DN3 thymocytes. Importantly, we showed that in contrast to N1, N3 has a low binding affinity for DL4, the most abundant intrathymic DL ligand. Thus, despite the profound effects of ectopic ligand-independent N3 activation on T cell development and leukemogenesis, physiologically activated N3 is dispensable for both processes, likely because N3 interacts poorly with intrathymic DL4.

No late ulnar artery atheroma after radial artery harvest for coronary artery bypass surgery.

BACKGROUND: Radial artery harvest for coronary artery surgery leads to chronically elevated blood flow in the remaining ulnar artery. This study examined the ulnar artery for evidence of increased atherosclerosis compared with the contralateral ulnar artery where the radial artery had not been harvested. METHODS: Patients were enrolled at least seven years after unilateral radial artery harvest. Anatomical and flow data were acquired using a high-frequency ultrasound probe. Maximal forearm blood flow was measured after repeated hand grip with concurrent brachial artery occlusion to induce forearm ischemia. RESULTS: Eighty five patients, 71 males at age 71 +/- 9 years (43 to 88) were assessed at 8.4 +/- 1.0 years (7.2 to 11.1). There was no patient with ulnar artery atheroma on either side. Mild ulnar calcification was present in four patients bilaterally. The ulnar diameter after radial artery harvest was greater (2.8 +/- 0.5 vs 2.4 +/- 0.4 mm; p < 0.001), as was flow at rest (111 +/- 64 vs 59 +/- 41 mL/min; p < 0.001). However, the brachial artery flow was not different between the two sides at rest (169 +/- 90 vs 176 +/- 87 mL/min; p = 0.060) or after ischemic exercise (714 +/- 294 vs 753 +/- 315 mL/min; p = 0.485). CONCLUSIONS: At an average of eight years after radial artery harvest, the remaining ulnar artery does not have evidence of increased atheroma and the maximal forearm blood flow is preserved.