Background
Thymus is the key organ for development and maturation of T lymphocytes. The dysfunction of thymus leads to decreased immunity or autoimmune diseases. T cells can be divided intoαβ T cells and γδ T cells, with distinct functions. αβ T cells and γδ T cells are derived from the same precursor cells, however, the molecular mechanisms underlying their differentiation were not completely clear.

The main research focus of professor Sun laboratory is to define the role of Cullin RING ligases (CRLs) and protein neddylation in regulation of tumorigenesis and other biological processes. CRL is the largest family of E3 ligase, consisting of multiple subunit including scaffold cullins. Neddylation modification is catalyzed by neddylation activating enzyme (E1), conjugating enzymes (E2, UBE2M and UBE2F) and ligase E3 (RBX1 and SAG/RBX2). The pair of UBE2M and RBX1 promotes neddylation of cullins 1-4, leading to activation of CRLs 1-4, whereas the pair of UBE2F and SAG promotes neddylation of cullin-5 to activates CRL5.

Research Progress
In this study, the SUN team focused on the regulatory role of the neddylation-CRLs system in the early development of T lymphocytes in thymus, using thymus specific conditional knockout mouse models. Specifically, thymus-specific knockout of either Ube2m, Ube2f, or Sag, did not cause visible abnormality in thymic development. However, Rbx1 deletion in the thymus led to severe thymic atrophy with reduction of thymic size to one-third of normal, delayed T cell development, and caused an abnormal increase in the proportion of γδ T cells. The results clearly demonstrated the important role of Rbx1 in the thymus development and the fate determination of αβ-γδ T cells.

As an ubiquitin ligase, Rbx1 catalyzes the ubiquitylation and degradation of many proteins. The pro-apoptotic protein Bim is one of known substrate of Rbx1-CRL4. Indeed, thymic deletion of Rbx1 led to the accumulation of Bim protein. To determine the causal role of Bim1 in thymic developmental defect, the team generated Rbx1 and Bim double knockout model and found a partial rescue of Rbx1 cKO phenotype in thymus, indicating a partial role of Bim in defective thymic phenotypes. Given the fact that a variety of key signal molecules are subjected to Rbx1 degradation, the core mechanism mediated by Rbx1 likely involves multiple pathways. Further study revealed that Rbx1 affects the development process of DN3 and DN4 T lymphocytes in the thymus, and enhances the generation of γδ T cells by regulating the Akt and NF-κB signals, as well as metabolic pathways (e.g. GSH metabolism).

Thymic γδ T cells include an immature Gzma-positive subpopulation. The single-cell RNA sequencing analysis revealed that Rbx1 deficiency impedes the maturation of γδ T cells, leading to an increased proportion of immature Gzma-positive γδ T cells in the thymus. This immature Gzma-positive γδ T cells can be further divided into three subpopulations, including a Ki67-positive proliferative subpopulation, and Rbx1 knockout reduced the proportion of this proliferative subpopulation. Rbx1 deficiency also altered the ratio between γδ T1 (interferon-γ secreting) and T17 (IL-17a secreting) subpopulations in thymus γδ T cells, implying that Rbx1 is involved in regulating the fate and functions of γδ T cells.

Future Prospects
This study demonstrates the pivotal role of Rbx1 in thymus development and fate determination of αβ-γδ T cells, as well as in regulation of γδ T cell functions, which may have potential to offer intervention strategies for diseases related to immunodeficiency or abnormal activation (such as autoimmune) by Rbx1 targeting.

The complete study is accessible via DOI: 10.34133/research.0774