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The transcription factor KLF2 regulates T cell trafficking by promoting expression of the lipid binding receptor, S1P1, and the selectin, CD62L. Recently, it was proposed that KLF2 also represses the expression of chemokine receptors. We confirm the upregulation of the chemokine receptor CXCR3 on KLF2 deficient T cells. However, we show that this is a cell nonautonomous effect, as revealed by CXCR3 upregulation on WT bystander cells in mixed bone marrow chimeras with KLF2 deficient cells. Furthermore, we show that KLF2 deficient T cells overproduce IL-4, leading to the upregulation of CXCR3 through an IL-4 receptor and eomesodermin dependent pathway. Consistent with the increased IL-4 production, we find high levels of serum IgE in mice with T cell specific KLF2 deficiency. Our findings support a model where KLF2 regulates T cell trafficking by direct regulation of S1P1 and CD62L, and restrains spontaneous cytokine production in naive T cells.
Kruppel-Like factors (KLFs) are a family of zinc-finger transcription factors that are expressed in a broad range of tissues and at various times in ontogeny (Pearson et al., 2008). Germline knockout of one of these factors, KLF2, is not compatible with life because of vascular defects (Kuo et al., 1997a; Lee et al., 2006). Studies done with KLF2 deficiency limited to only hematopoietic cells reported a striking loss of T cells from the blood, lymph node and spleen with thymic development appearing grossly normal (Kuo et al., 1997b).
Our laboratory previously reported an increase of mature CD4 and CD8 single positive (SP) cells in the KLF2 deficient thymus (Carlson et al., 2006). KLF2 deficient SP thymocytes survived in vitro and in vivo so that the lack of peripheral T cells is seemingly not a result of cell death (Carlson et al., 2006; Sebzda et al., 2008). Thus, the accumulation of mature SP cells in the thymus implied an emigration defect. Consistent with this, KLF2 deficient T cells showed severely reduced S1P1 expression (Carlson et al., 2006). S1P1 is a cell surface receptor for the phospholipid sphingosine-1-phosphate (S1P) and is required for thymic emigration (Mandala et al., 2002; Matloubian et al., 2004). KLF2 directly binds to the S1P1 promoter and induces S1P1 transcription (Bai et al., 2007; Carlson et al., 2006). KLF2 also regulates T cell expression of L-selectin (CD62L) (Bai et al., 2007; Carlson et al., 2006; Dang et al., 2009; Sebzda et al., 2008). Although CD62L is not required for thymic emigration, it is required for entry into lymph nodes (Arbones et al., 1994), and S1P1 is required for egress from lymph nodes (Matloubian et al., 2004). Thus, KLF2 acts as a single transcription factor controlling two key molecules—S1P1 and CD62L—required for naïve T cell trafficking through secondary lymphoid organs (SLO).
A recent report found that CD4 positive T cells from KLF2 deficient mice expressed multiple inflammatory chemokine receptors, suggesting that loss of KLF2 leads to redirection of naïve T cells to non-lymphoid sites (Sebzda et al., 2008). Together these findings leave us with the appealing idea that KLF2 acts as a master regulator of naïve T cell trafficking. KLF2 would direct naïve T cells through SLOs by positively regulating CD62L and S1P1, and would negatively regulate inflammatory chemokine receptors to prevent naïve T cells entering nonlymphoid tissues. However, in this report we demonstrate that expression of the chemokine receptor CXCR3 in KLF2 deficient T cells is regulated via a cell-nonautonomous pathway. We find that KLF2-knockout T cells exhibit dysregulated IL-4 production, which can act on bystander wild type T cells to induce aberrant expression of CXCR3. These data suggest KLF2 enforces naïve T cell trafficking by both autonomous and nonautonomous mechanisms. Furthermore, it suggests that KLF2 also maintains naïve T cell identity in terms of cytokine production, as KLF2 deficient T cells rapidly produce IL-4, a property usually associated with innate-immune and memory T cells.
To further study how KLF2 regulates chemokine receptor expression, we employed mice with a T cell specific deficiency in KLF2. We used CD4-cre mice crossed to mice with KLF2 flanked by loxP sites (KLF2fl) (Odumade et al.). In this model, the KLF2 gene is excised at the DN4/DP stage of thymocyte development prior to the SP stage where KLF2 is normally first expressed. Such mice have a similar T cell phenotype to KLF2 deficient fetal liver chimeras (Carlson et al., 2006) and to Vav-Cre/KLF2fl/fl mice (Sebzda et al., 2008). This includes severe peripheral T cell lymphopenia and a two-fold accumulation of mature SP thymocytes (Odumade et al.).
We compared chemokine receptors on SP thymocytes purified from WT versus CD4Cre/KLF2fl/fl mice (here on referred to as “KLF2 KO”). Although most SP thymocytes are conventional αβ T cell precursors, this population normally also includes low numbers of NKT, γδT cells, Treg, and recirculating memory T cells. Our lab previously showed that such nonconventional T cells can complicate the analysis of SP thymocytes (McCaughtry et al., 2007). To focus our analysis on conventional αβ T cells, we used a “dump strategy” to exclude cells that expressed CD25, TCRγ, NK1.1, or were capable of binding CD1d/αGal-cer tetramers. We observed a striking overexpression of CXCR3 mRNA in KLF2 deficient conventional thymocytes (Figure 1A), ten-fold for CD4SP and 40-fold for CD8SP compared to WT. CXCR3 protein expression was confirmed by flow cytometry and was observed both on thymocytes (Figure 1B) and peripheral T cells (data not shown). CXCR3 is typically not expressed on naïve cells, but can be upregulated on effector and memory T cells. It has three ligands: CXCL9, CXCL10, and CXCL11, which are produced at sites of inflammation. In contrast to a previous publication (Sebzda et al., 2008), we did not observe upregulation of any other inflammatory chemokine receptor mRNA, nor could we confirm protein upregulation for CCR3, CCR5, or CCR6 (Figure 1B and data not shown). While we cannot fully explain the discrepancies at this time, there are two key differences between the studies. First, Sebzda et al. used vav-cre while we used CD4-cre to delete KLF2. The other is our use of the dump strategy to exclude non-conventional T cells, as such cells are dysregulated in KLF2 deficient mice (Figure S1). Nonetheless, CXCR3 protein was clearly upregulated on conventional αβ T cells from CD4Cre/KLF2fl/fl mice.
During our study of CXCR3 on KLF2 deficient T cells, we noticed variation in the expression of CXCR3. In particular, CD8 T cells often expressed more CXCR3 than CD4 T cells, and the percentage of KLF2 deficient T cells expressing CXCR3 increased with age. In contrast, direct KLF2 targets—CD62L and S1P1—were consistently downregulated in KLF2 deficient T cells, independent of age (data not shown). We hypothesized that an altered thymic environment induced by KLF2 deficiency might lead to the increased CXCR3. To investigate the possibility of a bystander effect caused by KLF2 deficient cells, we set up mixed bone marrow chimeras where KLF2 deficient progenitors were a majority and WT cells were a minority (Figure 2A). Allelic markers, Thy1.1 and CD45.2, were used to distinguish donor populations from each other, and from host cells. Gating on CD4SP thymocytes from such chimeras, CD62L expression was reduced only in KLF2 deficient cells (upper left panel figure 2B), and not in WT progenitors (upper right panel figure 2B), confirming direct regulation by KLF2. As expected, CXCR3 was upregulated on KLF2 deficient thymocytes, and in chimeras of this age the CXCR3 upregulation was more striking on CD8SP than CD4SP (lower left panels figure 2B). Clearly, CXCR3 was also expressed on WT thymocytes in the predominantly KLF2 deficient thymus (lower right panels figure 2B), again more so on CD8SP than CD4SP. The expression of CXCR3 on WT cells indicates that KLF2 deficiency in T cells leads to cell extrinsic upregulation of CXCR3 on bystander cells.
It would still be possible that CXCR3 might be both directly and indirectly regulated by KLF2. However, in chimeras in which WT cells predominate, KLF2 deficient thymocytes did not express any detectable CXCR3 (Figure S2), suggesting that CXCR3 is only indirectly regulated by KLF2. CXCR3 was upregulated at the mRNA level in the WT bystander cells from the KLF2 deficient environment (Figure 3). In contrast, S1P1 mRNA levels were dramatically lower in KLF2 deficient T cells than in WT bystander cells (Figure 3) confirming direct regulation by KLF2. Thus, it would appear that KLF2 does not directly repress chemokine receptors, but its deficiency leads to upregulation of CXCR3 on bystander cells.
The hypothesis that KLF2 directly represses CXCR3 would predict that KLF2 and CXCR3 would not be mutually expressed in vivo. However, our findings would suggest instead that KLF2-deficiency has an indirect effect on CXCR3 expression. To explore this concept further, we wanted to assess expression of KLF2 and CXCR3 in individual T cells. The lack of detection of KLF2 by flow cytometry with commercially available antibodies has not allowed these studies. To overcome this obstacle, we generated knock-in mice where GFP was inserted in-frame at the KLF2 translational start site in exon 1, creating a GFP-KLF2 fusion protein (Figure S3A). The fact that homozygous KLF2GFP reporter mice are viable and have normal numbers of CD4 and CD8 T cells (figure S3B) implies that the GFP-KLF2 fusion protein is functional, in lieu of the embryological lethality of KLF2 KO mice. Furthermore, these mice appear to be faithful reporters of KLF2 expression, as GFP was not expressed until the mature (Qa2 hi, CD69 low) stage of SP thymocyte development (Figures S3C-F), consistent with what was previously reported for mRNA analysis (McCaughtry et al., 2007).
Since thymocytes do not normally express CXCR3, we examined KLF2 and CXCR3 expression in peripheral T cells. Interestingly, memory T cells (CD44 high) show heterogeneity in KLF2 expression (Figure 4), consistent with the fact that KLF2 is downregulated during T cell activation, but can be upregulated again (Schober et al., 1999). In these memory phenotype cells, CD69 and KLF2 (GFP) are inversely correlated (Figure 4 middle panels). This is consistent with the upregulation of CD69 observed on KLF2 deficient T cells (Carlson et al., 2006) as CD69 and S1P1 have been show to antagonize each other on the cell surface (Shiow et al., 2006). Thus the inverse correlation between CD69 and KLF2 may represent a positive correlation between S1P1 and KLF2, as expected from mRNA analysis. We find that S1P1 surface expression was limited to KLF2 expressing (GFP+) thymocytes (Figure S3G). In addition, we found that S1P1 mRNA was expressed at greater than 40 fold higher level in GFP+ compared GFP-thymocytes (data not shown). An inverse correlation would also be expected if KLF2 directly repressed chemokine receptors, such as CXCR3. However, there was no correlation between KLF2 (GFP) expression and CXCR3 in either CD4 or CD8 memory phenotype T cells (Figure 4, bottom panel). Thus our data do not support direct repression of chemokine receptors by KLF2.
Despite the fact that KLF2 does not directly regulate chemokine receptors, it is possible that the cell-nonautonomous upregulation of CXCR3 in KLF2 deficient mice could be responsible for lymphopenia by sequestering T cells in non-lymphoid organs. If peripheral lymphopenia was solely due to inappropriate chemokine receptor expression, then we would expect that CXCR3-expressing WT T cells in mixed bone marrow chimeras would also be reduced in the periphery. Alternatively, if lymphopenia is due to cell-autonomous gene regulation in KLF2 deficient thymocytes (for example reduced S1P1), we would expect KLF2 deficient T cells to be preferentially absent in the periphery. To answer this question we analyzed recent thymic emigrants (RTE) in mixed bone marrow chimeras. For this analysis, a covalent label is injected directly into the thymus, and peripheral tissues are evaluated two days later for cells bearing the label. The mixed bone marrow chimera approach not only allows analysis of cells with and without KLF2 in the same animal, it eliminates variability due to differential marking from the intrathymic injections. KLF2 is not expressed until the SP stage in thymocyte development so we standardized each chimeric animal to the ratio of KO:WT in the DP population. The ratio of KLF2 KO:WT increased from 1 in DP thymocytes to approximately 2 in SP thymocytes, consistent with an increased retention of mature KLF2 deficient thymocytes (Figure 5 and (Carlson et al., 2006). In contrast, the KLF2 KO:WT ratio dramatically decreased in CD4 and CD8 RTE in peripheral lymphoid organs indicating a strong preferential emigration of WT cells. We also found that WT RTE predominated in the liver as well, arguing against the idea that KLF2 deficient T cells exit the thymus normally but are preferentially sequestered in non-lymphoid tissue (Sebzda et al., 2008). Thus our data support a model of cell-intrinsic lymphopenia in KLF2 deficient mice, due in large part to impaired emigration from the thymus.
We next wanted to define the molecular mechanism for the cell-nonautonomous upregulation of CXCR3 in the presence of KLF2 KO T cells. Since KLF2 deficient thymocytes are retained in the thymic medulla, one possibility is that “thymic crowding” could create a situation where CXCR3 is upregulated. Therefore, we examined S1P1 deficient thymocytes, which have a similar thymic emigration defect and retention of mature SP thymocytes (Allende et al., 2004; Matloubian et al., 2004). Both KLF2 and S1P1 deficient thymi had an increased proportion of mature (HSA low) SP thymocytes. However, only KLF2 deficient SP expressed increased CXCR3 (data not shown). Thus, it is unlikely that cell-nonautonomous effects are due to “thymic crowding”.
Another possible mechanism is that KLF2 deficient cells might secrete a factor into the thymic environment, which causes the cell-nonautonomous effect. Using microarray analysis, we found that KLF2 deficient CD4 SP thymocytes express more IL-4 mRNA compared to WT. We confirmed the IL-4 mRNA upregulation by real time PCR (Figure 6A). To determine if KLF2 deficient thymocytes had the ability to produce IL-4 protein, KLF2 deficient and WT thymocytes were stimulated directly ex vivo with phorbol 12-myristate 13-acetate (PMA) and ionomycin. Significantly more KLF2 deficient CD4 and CD8 SPs produced IL-4 under these conditions (Figure 6B). Again, nonconventional T cells, such as NKT and Treg, were excluded from this analysis. Since NKTs are known to rapidly produce IL-4, we created CD4-cre/KLF2fl/fl/CD1−/− mice to test if invariant NKT cells were the source (Smiley et al., 1997). We observed the upregulation of CXCR3 on KLF2 deficient SP thymocytes in the presence and absence of invariant NKT cells (Figure S4). This suggests that KLF2 deficiency increases the potential for rapid IL-4 production in conventional T cells.
To determine if more IL-4 is produced in vivo as well, we analyzed the level of IL-4 receptor α (CD124), since IL-4 signaling leads to increased surface expression of CD124 (Ohara and Paul, 1988). CD124 levels were modestly, but consistently upregulated on thymocytes from KLF2 deficient mice (data not shown). This was also the case for both bystander WT and KLF2 deficient thymocytes in mixed chimeras where KLF2 deficient cells were the majority (Figure 6C). Since IL-4 is a major regulator of Th2 differentiation (Ansel et al., 2006), we looked for systemic effects of increased IL-4 levels by analysis of serum IgE, a classical type 2 antibody (Coffman et al., 1986). KLF2 deficient mice had a striking 33-fold increase in serum IgE compared to age matched controls (Figure 6D). To distinguish a specific IgE upregulation from broad hypergammaglobulinemia we assayed a broader range of isotypes. IgM, IgA, IgG3, IgG2a and IgG2b were not significantly changed between the WT and KLF2 deficient mice. IgG1 was significantly but more modestly upregulated compared to IgE (Figure S5). This is consistent with IL-4’s known role in inducing IgE and IgG1 (Snapper and Paul, 1987). Thus, T cell specific KLF2 deficiency leads to elevated IL-4 in vivo and systemically elevated IgE in the serum.
To test if CXCR3 expression in the KLF2 deficient thymus was dependent on IL-4 responsiveness, we generated mixed chimeras where the majority was KLF2 deficient progenitors and the minority was either WT or IL-4Rα KO. Again, WT bystander cells in the KLF2 deficient environment expressed CXCR3. However, IL-4Rα KO bystander cells did not show CXCR3 upregulation suggesting they are resistant to this bystander effect (Figure 6E right panels). Importantly, the KLF2 deficient cells from the same thymus as the IL-4Rα KO cells did express CXCR3 (Figure 6E left panels). This indicates that CXCR3 induction is strictly dependent on IL-4.
IL-4Rα can pair with either the common γ chain to produce type I IL-4 receptor or pair with IL-13 receptor for the type II receptor (Ramalingam et al., 2008). In mixed chimeras, IL-13R KO bystander cells in a KLF2 deficient thymus still upregulated CXCR3 (Figure S6), suggesting that type II IL-4 receptors are dispensable. Hence, taken together, these data suggest the type I IL-4 receptor (IL-4Rα/common γ chain) is required for bystander T cell upregulation of CXCR3.
From past work, it was shown that CXCR3 is primarily induced during Th1 helper responses; yet IL-4 skews T cells toward a type 2 response (Zhu and Paul, 2008). Thus our finding of CXCR3 upregulation due to IL-4 was unexpected. However, the paradigm of CXCR3 association with Th1 cytokines was established in activated CD4 T cells, and in our model CXCR3 is expressed most strongly on naïve CD8 thymocytes. Hence, we directly tested the impact of IL-4 influencing CXCR3 expression on naïve CD8 T cells. Interestingly, in vitro culture of thymocytes and splenocytes showed that IL-4 (with or without IL-7), but not IL-7 alone, induced expression of CXCR3 but not CCR3, on CD8 SP thymocytes and mature T cells (Figure 6F and data not shown). Thus, we propose that KLF2 deficient T cells spontaneously produce IL-4, and this cytokine efficiently upregulates CXCR3 expression on both WT and KLF2 deficient naïve CD8 T cells.
CXCR3 expression in T cells is dependent on the T box transcription factors eomesodermin (eomes) and/or T-bet (Intlekofer et al., 2008; Lord et al., 2005). Again, our microarray analysis identified the upregulation of eomes mRNA on KLF2 deficient SP thymocytes. We confirmed that both KLF2 deficient CD4 and CD8 SPs have higher expression of eomes mRNA compared to WT by real time PCR (Figure 7A). While eomes was over-expressed, the highly homologous transcription factor T-bet was not (Figure 7A). Previous studies showed that Tc2 skewing, using IL-4, can induce eomes in antigen stimulated CD8 T cells in vitro (Takemoto et al., 2006). Thus, we wished to determine if eomes was required for the bystander CXCR3 upregulation in KLF2 deficient mice.
A T cell specific eomes deficient mouse strain has recently been described (Intlekofer et al., 2008), and these animals display normal thymic development. To probe the role of eomes in bystander CXCR3 induction, we again generated mixed chimeras comprising a majority of KLF2 deficient cells with a minority of WT or CD4-Cre Eomesfl/fl bone marrow. When KLF2 deficient thymocytes were the majority, they expressed CXCR3 (Figure 7B left panels). As expected WT bystander cells expressed CXCR3, however eomes deficient bystander cells did not upregulate CXCR3 (Figure 7B right panels). These data suggest that eomes is essential for the induction of CXCR3 in the bystander T cell population.
Together, these data suggest a model for CXCR3 upregulation in the KLF2 deficient thymus, as summarized in Figure S7. In the KLF2 deficient thymus, bystander CXCR3 upregulation is dependent on IL-4 derived from KLF2 deficient cells, which signals via the type I IL-4R pathway acting through the transcription factor eomesodermin.
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/aGal-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/CD1−/− 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.
C57BL/6 (B6) and B6.SJL-Ptprca Pepcb (CD45.1 congenic B6) mice were purchased from the National Cancer Institute. B6.PL-Thy1a (CD90.1 congenic B6) and BALB/c-Il4ratm1Sz (IL-4 receptor KO) mice were purchased from Jackson Labs. C57BL/6NTac-Tg(CD4-cre) (Lee et al., 2001) were obtained from Taconic farms. KLF2fl mice were created in Jerry Lingrel’s laboratory at the University of Cincinnati and are described in a manuscript in preparation. In brief, loxP sites were inserted into introns 1 and 2 of the KLF2 allele. The KLF2 floxed mice were generated in Duffy ES cells (129SvEv/Tac) and backcrossed to the B6 strain for a minimum of 5 generations before being crossed with CD4-cre mice. Ingenious Targeting Labs generated the KLF2GFP reporter mice according to the strategy in figure S3A, using C57BL/6 ES cells. OT-I mice (C57BL/6 TCR transgenic strain specific for OVA257-263/Kb (Hogquist et al., 1994)) were maintained in our facility. All animal experimentation was conducted according to IACUC guidelines at the University of Minnesota.
Mixed bone marrow chimeras were generated by mixing T cell-depleted bone marrow preparations from Thy1 distinct, CD45.2+ strains, at various ratios and injecting 5–10x106 total cells into lethally irradiated (1000 rads) CD45.1+ host animals. For chimeras with Eomes KO and IL-13R KO bone marrow, femurs and tibias were shipped overnight on wet ice in RPMI + 10% FCS from Steve Reiner’s laboratory (University of Pennsylvania) and Thomas Wynn’s laboratory (NIAID), respectively. For chimeras with IL-4R KO bone marrow, the recipients were treated with NK1.1 specific antibody (clone PK136) intraperiotinally 50 μg one day prior then 25 μg days 7 and 14 after the marrow transplant to deplete NK cells and prevent graft rejection, as the IL-4R KO and control marrow were BALB/c origin and the recipients were C57BL/6 x BALB/c F1 mice. All chimeras were analyzed 8–12 weeks post transplant. Single cells suspensions of thymus, lymph node, and spleen were stained with FACS antibodies and analyzed by flow cytometry.
Single cell suspensions from spleen, lymph nodes, or thymus were prepared. Biotinylated CD1d-αGalCer monomers (NIH tetramer facility) were incubated with Streptavidin-PE or Streptavidin-APC for at 4° C overnight to create fluorescent multimers. Cells were analyzed on Becton Dickinson a LSR II instrument and the data was processed using FlowJo (Tree Star) software. Antibodies to standard mouse lymphocyte surface antigens were purchased from Biolegend (San Diego, CA) or eBioscience (San Diego, CA). In addition, antibodies to CXCR3-PE (R&D Systems (Minneapolis, MN), CCR3-AF647, CCR5-biotin, and CD124-biotin (all from BD Pharmingen, San Diego, CA), were used. A polyclonal antibody to S1P1 was the kind gift of Jason Cyster (University of California, San Francisco) and has been described previously (Lo et al., 2005). For detection, anti-rabbit IgG bioin (BD Pharmingen) was used followed by Streptavidin-APC (Invitrogen, Carlsbad, CA).
Fluorescence-activated cell sorting (FACS) was used to purify, “dump” negative CD4SP and CD8SP. To purify the minority population from mixed chimeras, the majority population was depleted by negative selection with anti-CD90.2 microbeads using MACS separation columns (Miltenyi Biotech, Bergisch Gladbach, Germany). Sorting was performed on a FACSAria (Becton Dickinson) and was reliably >90% of target population. RNA was isolated from sorted populations using the RNeasy kit (Qiagen, Valencia, CA) and cDNA was produced using the SuperScriptIII Platinum Two-Step qRT-PCR kit (Invitrogen), PCR products were amplified using Fast Start SYBR Green Master mix (Roche, Basel, Switzerland), and detected using a SmartCycler (Cepheid, Sunnyvale, CA). Hypoxanthine-Guanine Phosphoribosyl Transferase (HPRT) was used to normalize samples. Each group was sorted in at least two independent experiments and cDNA was prepared twice from each sort.
Primers were designed using the Ensembl database and Primer3. Primers are published in the supplement to this paper.
Sedated mice were intrathymically injected with up to 10 μL/lobe of 5 mg/mL sulfo-NHS-LC biotin from Pierce Chemical Co. (Rockford, IL) in PBS. 48 hours later, thymus, lymph node, spleen, and liver were harvested and stained with fluorescently conjugated streptavidin and other antibodies and analyzed by flow cytometry. Analysis of streptavidin binding on splenic B cells was routinely performed to ensure that covalent labeling was restricted to the thymus.
Thymocytes were isolated and plated at 1x106 cells/mL. The cells were stimulated with 50 ng/mL PMA and 1.5 μM ionomycin both from (Sigma-Aldrich, St. Louis, MO) for five hours with GolgiStop Protein Transport Inhibitor (BD Pharmingen) added for the final three hours. The cells were surface stained then stained with anti-IL-4 (11B11) (eBioscience) using a fix/perm kit (BD Pharmingen) and analyzed by flow cytometry.
A mouse IgE ELISA MAX kit (Biolegend) was used to quantify IgE from serum samples. Samples were run in duplicate.
Splenocytes and thymocytes from OT-I mice were cultured (1 x 106 splenocytes/ml, 5 x 106 thymocytes/ml) with IL-7 (10 ng/ml), IL-4 (20 ng/ml) or both for 4 days. Cells were analyzed by flow cytometry using an anti-CXCR3-PE (220803) antibody. Cytokines and antibody were purchased from R&D Systems.
Statistical analysis was performed using Prism (Graphpad software) unpaired T tests were used for IgE data and paired T tests were used for RT PCR and normalized RTE analysis.
The authors wish to thank Caitlin Dejong for preparing and shipping eomes KO bones, and Thirumalai Ramalingam and Thomas Wynn for providing IL-13R KO bones. Jerry Lingrel provided KLF2fl mice. Jason Cyster provided reagents and assistance with anti-S1P1 staining. Xiao Jie Ding and Jason Vevea provided excellent technical support. We thank Oludare Odumade and Amy Moran for helpful discussion and critical review of the manuscript. The authors declare no competing financial interests. This research was supported by NIH grants R01-AI39560 (to K.A.H.), R01-AI38903 (to S.C.J.) and T32-AI007313 (to M.A.W.).
Supplemental Data: Five figures of supplemental data can be found at:
AUTHORSHIP: M.A.W. designed and performed research and drafted the manuscript, K.T. and C.S. performed research, S.L.R. provided vital reagents, S.C.J., and K.A.H. designed research, interpreted data and edited the manuscript.
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