Gfi1

Regulatory T cells (T_reg), defined by the expression of the transcription factor Foxp3, are crucial for immunological self-tolerance and homeostasis.¹ T_reg cells are mainly developed in the thymus as natural T_reg cells (nT_reg), which can be best depicted by a 2-step model. The initial signals from the high-affinity T-cell receptor (TCR) and CD28 co-stimulation lead to the generation of a precursor population with enhanced cytokine responsiveness, which then responds to signals from γc-dependent cytokines, especially IL-2 for full induction of Foxp3 expression².

Consistent with the importance of TCR and co-stimulatory signals for nT_reg development, several transcription factors downstream of TCR/CD28 have been implicated in Foxp3 expression². For instance, among TCR-activated NFκB members, c-Rel has a crucial role in Foxp3 induction by binding to an intronic conserved noncoding sequence (CNS) in the Foxp3 locus, known as Foxp3-CNS3.² Concurrent with transcriptional activation of Foxp3 expression, TCR stimulation also leads to epigenetic modification by establishing a T_reg-specific CpG hypomethylation pattern in Foxp3-CNS2, as well as in other T_reg signature genes.³ Both transcriptional activation of Foxp3 expression and establishment of T_reg-specific epigenetic modifications are required for a full developmental and suppressive program of nT_reg cells. Aside from TCR/CD28 stimulation, IL-2 directly contributes to Foxp3 transcription by activating Stat5, and thus is intimately linked to nT_reg development.² IL-2 is also required for functional programming and expansion of nT_reg cells during thymic development.⁴ While remarkable advances have been made in deciphering TCR- and IL-2-induced intrinsic events in nT_reg development, how the production of extrinsic signals such as IL-2 is controlled remains poorly understood.

We addressed this issue by focusing on growth factor independent 1 (Gfi1), a transcription repressor important for immune and hematopoietic cell development and function. Since Gfi1 has been implicated in early thymocyte development,⁵ we utilized a mouse genetic model with T cell-specific deletion of Gfi1 via CD4-Cre (Gfi1^CD4-Cre) to circumvent the early developmental defects associated with germ-line deletion of Gfi1. We identified a previously unappreciated mechanism in which Gfi1 exerts a non-cell-autonomous effect to curtail nT_reg development by inhibiting IL-2 production from conventional T cells⁶ (Fig. 1). Combining pharmacological blockade of IL-2 signaling and genetic deletion of Il2, we showed that excessive IL-2 production in Gfi1^CD4-Cre mice underlined the augmented nT_reg development in Gfi1^CD4-Cre mice. Furthermore, when wild-type bystander cells were introduced into a Gfi1^CD4-Cre-predominant environment created by bone marrow chimeras, they exhibited an increased proportion of Foxp3⁺ nT_reg cells, indicative of an nT_reg-inducing environment established by Gfi1-deficient thymocytes. This non-cell-autonomous effect of Gfi1 on nT_reg development was further supported by the lack of effect on nT_reg development when Gfi1 was deleted selectively in T_reg cells. Importantly, the increased generation of nT_reg cells in the absence of Gfi1 was independent of its pro-survival effect. Taken together, Gfi1-mediated transcriptional control of extrinsic signals (IL-2) orchestrates an unconventional mechanism for nT_reg development, thereby complementing the well-recognized T_reg-intrinsic transcriptional pathways.

Figure 1. Control of Foxp3⁺ nT_reg cells and antitumor immunity by Gfi1. Loss of Gfi1 in conventional T cells (T_conv) leads to increased secretion of IL-2, which promotes nT_reg cell expansion in the thymus and periphery. Gfi1 deletion also upregulates T_reg effector molecules CD103 and CD73. Collectively, Gfi1 deficiency in T cells dampens antitumor immunity. ↑, increased expression or cellularity.

Deficiency of Gfi1 not only led to increased generation of nT_reg cells, but also potentiated their suppressive activity.⁶ Because tumor-infiltrating T_reg cells have a crucial role in dampening antitumor immunity, we examined whether the increased T_reg cellularity and function in the absence of Gfi1 impaired antitumor immunity. When challenged with B16 melanoma cells, Gfi1^CD4-cre mice were unable to control late-stage tumor growth, associated with significantly elevated T_reg/non-T_reg ratios. Moreover, Gfi1-deficient nT_reg cells showed increased expression of T_reg effector molecules CD103 and CD73 under steady-state and in the tumor environment. The integrin molecule CD103 marks the subset of T_reg cells with elevated suppressive activity in various contexts, including antitumor responses, whereas the ectonucleotidase CD73 is important for converting the proinflammatory extracellular ATP to immunosuppressive adenosine. Therefore, Gfi1 deficiency in T cells established a tumor-supportive environment by both increasing T_reg cellularity in tumors and endowing them with a greater suppressive activity via upregulating CD103 and CD73 expression. Mechanistically, direct inhibition of CD103 and CD73 by Gfi1 has been reported in induced T_reg cells⁷ and T_H17 cells,⁸ respectively. Our results support and extend these findings by showing that Gfi1-deficient nT_reg cells express elevated levels of CD103 and CD73.⁶ It is also noteworthy that the effect of Gfi1 deficiency on CD103 upregulation appeared to be less pronounced in the absence of IL-2 (our unpublished findings), suggesting that enhanced IL-2 production in the unmanipulated Gfi1^CD4-cre thymus also contributes to CD103 upregulation. This interpretation is consistent with the effect of IL-2 to upregulate CD103 and CD73 expression for functional maturation of thymic nT_reg cells.⁴ We therefore propose that Gfi1 deficiency upregulates CD103 and CD73 expression in nT_reg cells via both direct and indirect mechanisms, thereby potentiating nT_reg-mediated immunosuppression in Gfi1^CD4-Cre mice.

In conclusion, Gfi1 orchestrates an important cell-extrinsic control mechanism for nT_reg development by regulating IL-2 production from conventional T cells. Further, Gfi1 likely exerts both extrinsic and intrinsic effects on nT_reg-suppressive activity by mitigating IL-2 production from conventional T cells and by directly regulating nT_reg effector molecules, respectively. Given the extensive effects of Gfi1 deficiency on nT_reg development and function, strategies to enhance Gfi1 expression and/or activity represent a possible approach for antitumor immunotherapy.

Shi LZ, et al. Proc Natl Acad Sci U S A. 2013;110:E3198–205. doi: 10.1073/pnas.1300950110.

10.4161/cc.26824