Our data provide compelling evidence for a cell-nonautonomous or bystander effect leading to chemokine receptor upregulation in the KLF2 deficient thymus. There is precedence for cell-nonautonomy in many gene deficient mouse models (Whyatt and Grosveld, 2002
) and in C. elegans
(Apfeld and Kenyon, 1998
). Cell-nonautonomous effects have been previously observed in the thymus as well (Schnell et al., 2006
). When tissue specific promoters are used to direct gene deficiency to one cell type, and effects are observed on another cell type, it is easy to conclude that cell-nonautonomous effects are involved. In our case, however, T cell specific gene deletion caused cell-nonautonomous effects on other T cells, which could only be revealed by analysis of chimeric mice where both WT and gene-deleted cells were present in the same population.
The paucity of KLF2 deficient T cells in SLOs and blood has been a consistent finding in all models studied, and published studies agree that KLF2 regulates multiple factors important for T cell migration. But what molecules normally regulated by KLF2 cause the peripheral lymphopenia? Sebdza and colleagues suggested that KLF2 deficiency de-represses multiple chemokine receptors leading to tissue localization of T cells (Sebzda et al., 2008
). We think this is unlikely to account for the lymphopenic phenotype for several reasons. First, the lack of S1P1
is profound. We were unable to detect S1P1
staining above background on CD4SP or CD8SP. KLF2 and S1P1
T cell deficiency cause a similar accumulation of mature thymocytes (Carlson et al., 2006
; Matloubian et al., 2004
). In both cases there is a significant (two fold) increase in the number of mature SP thymocytes, which is inconsistent with normal thymocyte emigration. One distinction is the CD62L high phenotype of T cells with S1P1
deficiency, whereas KLF2 deficient T cells have low CD62L (Matloubian et al., 2004
). The lack of CD62L on KLF2 deficient T cells is consistent with direct regulation by KLF2 (Bai et al., 2007
; Dang et al., 2009
). Since CD62L is necessary for lymph node entry (Arbones et al., 1994
), this also explains differences in trafficking between KLF2 and S1P1
deficient T cells when adoptively transferred into the blood (Carlson et al., 2006
; Matloubian et al., 2004
). Secondly, we did not observe preferential homing of KLF2 deficient T cells to the liver, in contrast to a previous report (Sebzda et al., 2008
). Finally, we observed the upregulation of only one chemokine receptor (CXCR3) rather than an increase in 10 chemokine receptors observed by Sebzda et al. These differences may all relate to the model systems used. Sebzda et al. induced KLF2
deletion via Cre driven by the Vav promoter (Sebzda et al., 2008
), which is expressed in all hematopoietic cells (Stadtfeld and Graf, 2005
), while our system (using CD4-Cre) has expression limited to the T cell lineage (Lee et al., 2001
). Furthermore, our analysis was focused on “conventional” T cells, and we excluded cells expressing CD25, TCRγ, NK1.1, and capable of binding CD1d/a
Gal-cer tetramers. We are currently exploring the role of KLF2 in the homeostasis and migration of nonconventional T cell subsets to address this point further.
While our findings do not support a model in which KLF2 directly represses chemokine receptors, they led us to discover a novel function for KLF2: the cell-intrinsic suppression of IL-4 in T cells. KLF2 deficient thymocytes had increased levels of IL-4 mRNA, and were able to rapidly produce the cytokine upon stimulation ex vivo
. Elevated serum IgE levels suggested that IL-4 is overproduced in vivo
as well. NKT cells are major, rapid producers of IL-4. One possibility for the increased IL-4 in the KLF2 deficient thymus is dysregulated homeostasis, trafficking and/or activation of NKT cells. To address this, we generated CD4-cre/KLF2fl/fl
mice, in which deletion of CD1 drastically reduces the number of NKT cells (Smiley et al., 1997
). The thymocytes from these mice still had increased expression of both CXCR3 and CD124 indicating that IL-4 continued to be overproduced. This suggests that invariant NKT cells are not the sole or major provider of spontaneous IL-4 and the bystander effect in this model. We favor the hypothesis that naïve, conventional αβ T cells, which normally have high KLF2 expression and low capacity to produce IL-4, rapidly produce IL-4 in the absence of KLF2. Whether the effect of KLF2 acts directly or indirectly in regulating the IL-4 locus is currently unclear. IFNγ and TNFα were also overproduced by KLF2 deficient T cells (data not shown). Thus, it is interesting to note that KLF2 deficient “naïve” T cells exhibit some functions similar to “innate immune” T cells.
Overall, our findings suggest KLF2 is an important transcriptional regulator of naïve T cell identity in that it promotes the ability of T cells to recirculate through secondary lymphoid organs, while at the same time repressing rapid cytokine production in the naïve T cell pool.