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Kruppel-like transcription factors are a large family of proteins that can both activate and repress genes and regulate a wide variety of biological processes in multiple organ systems. In T lymphocytes, Kruppel-like factor 2 (KLF2) is an essential gene, as very few T cells are found in the spleen and lymph nodes of mice with targeted deficiency of KLF2 1. This was initially thought to reflect a requirement for KLF2 in T cell survival and homeostasis. However, subsequent studies showed that KLF2 was required for thymic emigration and lymph node homing by regulating cell surface receptors required for these processes, namely S1P1 and CD62L 2. Now Mark Kahn’s group adds a new twist to the story. They show that KLF2 also represses chemokine receptor gene expression 3. Thus KLF2 deficient T cells aberrantly express multiple chemokine receptors that can cause T cells to home to various tissues in the body. Altogether, these studies establish KLF2 as a “master regulator” that coordinates expression of multiple different types of cell surface receptors to control T cell trafficking during an immune response.
Sedbza and colleagues employed a VAV-Cre transgene to eliminate KLF2 in lymphocytes 3. Their results confirmed that there is not a substantial survival defect in KLF2-null (KLFo) T cells due to spontaneous apoptosis. They further confirmed that two genes critical for T cell trafficking, S1P1 and CD62L, are down regulated in T cells lacking KLF2, and that these mice have increased numbers of mature thymocytes [AU: delete “in the thymus”? by definition thymocytes are in the thymus?] and reduced numbers of peripheral T cells in the blood, lymph node, and spleen. Surprisingly however, they report a 3-5-fold increase in the number of KLF2 deficient T cells outside of lymphoid organs. This was inferred by assessing CD4 mRNA by real-time PCR in various tissues, such as bone marrow, liver, kidney, muscle and brain. Indeed, they used flow cytometry to confirm a 3-fold increase in the number of KLF2o CD4 T cells in the liver. To explain this altered migration pattern, they examined chemokine receptor expression, and found an increase in multiple chemokine receptors that generally control cellular movement to various inflamed and non-inflammed tissues, suggesting that KLF2 is a general repressor of certain chemokine receptors.
Together, these data clearly point to KLF2 as a critical regulator of T cell circulation patterns (Figure 1). KLF2 activates two genes that control access to, and emigration from, lymph nodes: CD62L and S1P1. As such, naïve T cells, which express KLF2, hold the keys to the front and back door of the lymph node—and primarily circulate through secondary lymphoid organs (red arrows). When this mode of trafficking is “on”, it would seem important that the expression of chemokine receptors that direct T cells to peripheral sites be “off”. Thus it makes intuitive sense that the same transcriptional regulator would also repress certain chemokine receptors. Indeed, when T cells become activated, they rapidly extinguish expression of KLF2, losing S1P1 and CD62L, and inducing chemokine receptors that direct the T cell to peripheral tissues and sites of inflammation (shaded blue). If the mature T cell becomes activated in the thymus, it likewise is retained there 4 These data thus solidify the notion that the critical role of KLF2 is to coordinate multiple molecules (selectins, chemokine receptors, and sphingolipid receptors) that need to act together to for appropriate cellular trafficking. (Figure 1).
It is tempting to speculate that KLF2 controls these multiple genes by directly binding to the promoters and recruiting co-factors for activation or repression. Indeed, KLF2 was shown to directly bind the S1P1 promoter and could transactive expression of a reporter gene with an isolated S1P1 promoter 2. Similar transactivation studies were performed with CD62L5 and both of these genes have a KLF2 consensus binding site in their promoters. Sedbza et al. showed that KLF2 could transactivate reporter constructs with CCR3 and CCR5 promoters, suggesting that KLF2 directly represses these genes. It will be important to establish whether this is the case for other chemokine receptors.
An interesting suggestion arising from the study by Sebzda et al. is that KLF2 may not be required for thymic emigration per se, but rather that deficient T cells leave the thymus normally and are immediately sequestered in non-lymphoid tissues. Consistent with this, the authors showed that recent thymic emigrants (RTE) could be recovered from the liver of KLF2 deficient mice. Even presuming RTE are also present in other tissues, it is difficult to know if the increased number of RTE in tissues could numerically represent “normal” thymic emigration (1–2 × 106/day). The idea that thymic emigration is not impaired in KLF2o mice is difficult to reconcile with other data, as well. For example KLF2o SP thymocytes are overrepresented in the thymus. Such retention would not be predicted if emigration occurred normally in KLF2o mice. In fact, using a RAG2GFP reporter as a “molecular timer” we found that KLF2o mature thymocytes are retained for a striking length of time (at least 5 times longer than wt cells; unpublished data[AU: pls insert appropriate reference citation here]). Normal thymic emigration is also inconsistent with the profound reduction of S1P1 mRNA and protein in KLF2o T cells 2,3. The authors suggest that KLF2o T cells, despite a dramatic reduction of message and cell surface protein, may have enough residual S1P1 to emigrate normally. This is based on their finding that an S1P agonist, FTY720, could influence migration of KLF2o T cells. However, it should be noted that FTY720 binds to three other S1P receptors, of which at least one, S1P4, is known to be expressed in T cells 6. Further work will be required to more precisely quantitate the emigration defect and accumulation of KLF2o T cells in tissues. Nonetheless, the current findings clearly reveal an important new set of KLF2 target genes.
Since KLF2 is emerging as a critical regulator of T cell trafficking, it is imperative to understand what signals regulate KLF2 expression itself. T cell receptor signaling is clearly important, as KLF2 mRNA is dramatically repressed after T cell activation. KLF2 protein may also be de-stabilized by phosphorylation and targeted for degration 7. In endothelial cells KLF2 is regulated via the map kinase ERK5 and the transcription factor MEF2D 8. However, it is not known if this pathway is involved in regulatign the expression and function of KLF2 in lymphocytes. Interestingly, in the thymus, KLF2 seems to be upregulated by TCR signaling, in the sense that it is not expressed until after positive selection 1. However, a recent study showed that KLF2 is not upregulated directly after positive selection, but rather at the latest mature SP stage 9. We estimate this is at least 3 days after the onset of positive selection signaling. Thus it seems likely that there are other developmental or microenvironmental signals that direct the cell to express KLF2, and subsequently emigrate from the thymus, and adopt the circulation pattern of a naïve T cell. It will also be interesting to determine how KLF2 is regulated in memory T cells, and whether it controls the balance of central and effector memory cells. Ultimately, manipulation of KLF2 in peripheral T cells may be a promising way to control lymphocyte migration patterns for therapeutic purposes.