Co-stimulation regulates T cell activation, but it remains unclear whether co-stimulatory pathways also control T cell differentiation. We used mass cytometry to profile T cells generated in the genetic absence of the negative co-stimulatory molecules CTLA-4 and PD-1. Our data indicate that negative co-stimulation constrains the possible cell states that peripheral T cells can acquire. CTLA-4 imposes major boundaries on CD4⁺ T cell phenotypes, whereas PD-1 subtly limits CD8⁺ T cell phenotypes. By computationally reconstructing T cell differentiation paths, we identified protein expression changes that underlied the abnormal phenotypic expansion and pinpointed when lineage choice events occurred during differentiation. Similar alterations in T cell phenotypes were observed after anti-CTLA-4 and anti-PD-1 antibody blockade. These findings implicate negative co-stimulation as a key regulator and determinant of T cell differentiation and suggest that checkpoint blockade might work in part by altering the limits of T cell phenotypes.

共刺激调节T细胞活化，但是尚不清楚共刺激途径是否也控制T细胞分化。我们使用大量的细胞计数法来分析在没有负共刺激分子CTLA-4和PD-1的遗传情况下产生的T细胞。我们的数据表明负共刺激限制了外周T细胞可能获得的细胞状态。CTLA-4在CD4 ⁺ T细胞表型上强加了主要界限，而PD-1巧妙地限制了CD8 ⁺T细胞表型。通过计算重建T细胞分化途径，我们确定了蛋白质表达的变化，这些变化是异常表型扩展的基础，并指出了分化期间发生谱系选择事件的确切时间。抗CTLA-4和抗PD-1抗体阻断后，观察到T细胞表型的类似变化。这些发现暗示负共刺激是T细胞分化的关键调节剂和决定因素，并暗示检查点封锁可能部分通过改变T细胞表型的限制而起作用。