Genetic Ablation of Cereblon (CRBN) Increases Long-Lived Memory T Cells

Background: Immunomodulatory drugs (IMiDs) consist of thalidomide and derivatives, pomalidomide and lenalidomide. IMiDs target the E3 ubiquitin ligase substrate receptor, cereblon (CRBN), which forms a complex with DDB1-Cul4A, to induce anti-proliferative effects in tumor cells and increase the activation of T cells. Using crbn deficient T-cells, we have shown that CRBN is a negative regulator of T cell activation in mice. Dynamic changes in the T-cell repertoire occur after thymic involution. With the reduction of thymic output with age, there is a loss of naïve T-cells in the peripheral blood, and the accumulation of a memory-like T-cell population that arises through homeostatic proliferation.  In this study, we demonstrate the role of CRBN in peripheral homeostatic regulation during aging. 

Methods: In this study, C57BL6 crbn -/- mice were housed in pathogen-free conditions to limit foreign antigen exposure. Splenocytes were stained with T cell markers to define various memory populations from 3, 8, 10 and 13 month old crbn-/- and age-matched wild-type mice. Expression of CD8, CD44, CD127 (IL-7 receptor) and KLRG1 were determined by flow cytometry. CD44-CD127+ KLRG1- CD8+ T cells were considered naive in this analysis. Consistent with previous reports, CD44+ CD127+ KLRG1- T cells represent long-term memory T-cells while CD44+CD127-KLRG1+ T cells are effector memory cells. To better understand age-related changes in the thymus, the thymus was dissected and the absolute number of thymocytes was determined by trypan blue staining.  Thymic subpopulations were defined using CD4 and CD8 as single positive, double positive, and double negative cells in 13 month old crbn -/- and WT mice. We further characterized double negative (DN) populations using CD44 and CD25 to define DN1, DN2, DN3, and DN4.

Results: Splenocytes in 3 and 8 month old crbn -/- and WT mice have no significant differences in CD44+ memory cells, naïve cells (p=0.7850 and p=0.5061) or recently activated cells [CD69+ or CD25+]. Changes related to thymic involution in WT mice become evident at 10 months of age. Crbn -/- mice exhibit a similar percentage and absolute number of total memory cells (p=0.8194) at 10 months of age, as defined by CD44 expression.  Evaluation of long-lived versus short-lived sub-populations of memory cells in these older mice showed that crbn deficiency is associated with significantly more CD44+ CD127+ KLRG1- CD8+ [long-lived] memory T cells compared to WT mice (p<0.001). Evaluation of the thymus revealed no difference in absolute numbers of thymocytes in younger mice.  Moreover, crbn deficient mice have normal distribution of SP, DP, and DN populations, with only slight changes in DN1, DN2, DN3, and DN4 subpopulations. Absolute numbers of thymocytes, however, were significantly higher in the crbn -/- mice at 10 months of age compared to age-matched controls (p=0.0182).

Conclusions: The fate of developing T cells is regulated by positive and negative selection in the thymus where the coordination and selection of self-tolerant repertoire is maintained. Age-related changes caused by thymic involution impacts the protective responses of these cells against foreign pathogens and tumor cells.  Our data show that the genetic germline depletion of crbn leads to an increase in long-lived memory T cells in naturally aged animals. Universally in cancer patients, and especially in MDS, the lymphocyte compartment is characterized by premature age-related changes due possibly to reduction in lymphopoiesis at the stem cell level or due to chronic antigen stimulation.  Given that the CD44+ CD127+ KLRG1- population is significantly higher in aged crbn-/- mice, these mice may be more resistant to viral infection and or development of malignancy. The mechanism responsible for this phenotypic difference could be due to cell intrinsic signaling or metabolic properties. However, a role for CRBN in thymic epithelial cells must also be considered given the use of germline deficient animals in this study.  Taken together, we have demonstrated that ablation of crbn results in an increase in long-lived CD8+ T cells during aging, which suggests that targeting CRBN and/or treating with lenalidomide may functionally improve memory T cell capacity in the elderly.