T-bet is highly expressed by NKp46+ ILC
ILC patrolling the intestine comprise distinct populations that secrete diverse arrays of cytokines. Although the role of T-bet has been investigated in T and NK cells, its impact on various ILC populations present in the gut has not been determined. To address this question, we first assessed T-bet expression in total CD3ε− siLP lymphocytes (siLPLs) by intracellular multicolor flow cytometry analyses.
We initially focused on the CD3ε−NK1.1high cell populations residing in the siLP because these cells resemble T-bet–expressing conventional NK cells present in other organs (spleen and liver). Based on CD127 expression, it is possible to define two different NK cell subsets in the lamina propria, herein denoted CD127+ and CD127− NK cells. Notably, T-bet was highly and homogeneously expressed in both populations (, top).
Figure 1. T-bet is differentially expressed in populations of gut innate lymphocytes. (A) T-bet expression was assessed in cell populations discriminated based on expression of NK1.1 and CD127 (IL-7 receptor α), Rorγt, NKp46, and CD4. These included (more ...)
Aside from NK1.1, conventional NK cells can also be identified by NKp46 expression in several lymphoid (bone marrow, spleen, lymph node, and thymus) and nonlymphoid (liver and lung) organs. In addition, in the intestine, a specific NKp46+ cell subset that expresses low levels of NK1.1 is known to be an important producer of IL-22 (NKp46+ ILC22). We therefore analyzed T-bet expression in these subsets. Within CD3−NK1.1low/−Rorγt+CD127+ cells, three populations can be discerned based on CD4 and NKp46 expression: NKp46+ ILC22 (CD4−NKp46+), CD4− ILC22 (CD4−NKp46−, a heterogeneous population comprising ILC22 and LTi cells), and LTi4 (CD4+NKp46−). As shown in (bottom), although T-bet was barely detectable in CD4− ILC22 and LTi4, it was highly expressed in NKp46+ ILC22. The mean fluorescence intensity (MFI) of T-bet staining is depicted in . The data indicate that NKp46+ ILC22 expressed T-bet at comparable levels of CD127+ and CD127− NK cells. These results raise the possibility that although NKp46+ ILC22 have similar requirements for development with other ILC22 populations, T-bet could play a selective role in this subset.
Profound reduction of NKp46+ ILC22 and CD127+ NK cells in the absence of T-bet
To elucidate the potential impact of T-bet in these ILC subsets, we assessed the various populations of gut innate lymphocytes in Tbx21−/−
mice by applying the same gating strategy used in . Despite the homogeneous expression of T-bet among all CD3ε−
mice showed a dramatic and selective reduction of CD127+
NK cells. In contrast, there were no significant differences in CD127−
NK cells, compared with WT mice (). When we analyzed CD127+
ILC populations, we found no difference in the number of CD4−
ILC22 or LTi4
cells. However, in agreement with the pattern of T-bet expression, we did note a striking and selective decrease in the number of NKp46+
ILC22 cells in T-bet–deficient mice (). It was possible though that T-bet was simply regulating NKp46 expression on this subset. We attacked this problem in two ways. First, we found that NKp46 expression on conventional NK cells was not affected by the absence of T-bet (unpublished data). Second, we enumerated ILC22 cells using markers independent of NKp46+
. ILC22 have been shown to be enriched in the Rorγt+
population of lamina propria cells (Sawa et al., 2010
and ). We observed a significant reduced proportion of this population of cells in Tbx21−/−
mice, independent of reliance on NKp46 expression (). In addition, Tbx21−/−
mice had normal numbers of CD45+
cells, which contain ILC2 and lacked T-bet expression (unpublished data). Collectively, these data argue that indeed, NKp46+
ILC22 cells are preferentially affected by the loss of T-bet.
Figure 2. NKp46+ ILC22 and CD127+ NK cells are dramatically reduced in Tbx21−/− mice. Lymphocytes were isolated from siLP of WT and Tbx21−/− mice and analyzed by flow cytometry. (A) The left panel shows representative dot plots and (more ...)
T-bet regulates differentiation and functions of NKp46+ ILC22 and gut NK cells
Consistent with their high levels of T-bet expression, NKp46+ ILC22 were dramatically reduced in Tbx21−/− mice. However, these cells were not completely absent. This afforded us the opportunity to determine the functional impact of T-bet in these cells. As noted above, terminal differentiation of NKp46+ ILC22 is characterized by acquisition of Rorγt expression. NKp46+ ILC22 were identified by focusing on CD3ε−NKp46+ and assessing CD127 and NK1.1 expression (). Rorγt expression was measured by intracellular staining of NKp46+ ILC22 (gated as CD127+NK1.1low/−) and, for comparison, CD127− and CD127+ NK cells, which do not express Rorγt. The absence of T-bet was associated with concomitant reduction of Rorγt in the residual NKp46+ ILC22 ().
Figure 3. Absence of T-bet impairs Rorγt expression and IL-22 production in NKp46+ ILC22. (A) Lymphocytes were isolated from siLP of WT and Tbx21−/− mice and the different CD3ε−NKp46+ populations (according CD127 and NK1.1 (more ...)
We next investigated whether IL-22 production was also affected in the residual NKp46+
ILC22 present in T-bet–deficient mice. As previously shown, in response to IL-23, NKp46+
ILC22 from WT mice produce high levels of IL-22 (Satoh-Takayama et al., 2008
; ). However, the residual population of NKp46+
ILC22 present in Tbx21−/−
mice showed a severe impairment in IL-22 production (). In contrast, we found that IL-23 efficiently induced IL-22 production in LTi4
regardless of whether the cells were from Tbx21−/−
or WT mice (). Because IL-22 can also be a product of Th17 cells, we also asked if T-bet expression was important for the production of this cytokine in CD4+
T cells. We found that naive CD4+
T cells cultured in presence of IL-6, IL-1β, and IL-23 produced IL-17 and IL-22 (Ghoreschi et al., 2010
; ). However, the absence of T-bet did not influence the proportion of CD4+
T cells that produced IL-22 under these conditions. Collectively, we interpret these data to indicate that the impaired production of IL-22 in NKp46+
ILC22 expression likely reflects a maturational block, although the possibility remains that remnant cells present in Tbx21−/−
mice might represent a distinct subset. Conversely, T-bet is not involved in the regulation of IL-22, in LTi4
, and Th17 cells.
Cell-autonomous role of T-bet in the regulation of NKp46+ ILC differentiation
To formally establish that the phenotype we observed in Tbx21−/− mice was the result of a direct cell-intrinsic role of T-bet in NKp46+ ILC22 development, we transplanted lethally irradiated recipient mice (CD45.1) with bone marrow from WT or Tbx21−/− mice (CD45.2). After 8 wk, we found restoration of the various ILC22 subsets present in the lamina propria in mice transplanted with WT bone marrow (). In mice transplanted with T-bet–deficient bone marrow, we noted reconstitution of LTi4 cells; however, we observed that NKp46+ ILC22 cells were not present ().
Figure 4. Intrinsic role of T-bet in the regulation of ILC22 and NK cells. (A) Lethally irradiated WT CD45.1+ mice were transplanted with bone marrow cells from WT (CD45.2+) or Tbx21−/− (CD45.2+) mice. After 8 wk, siLPLs were isolated and innate (more ...)
To examine the effect of T-bet deficiency in a competitive environment, we next generated mixed bone marrow chimeras, transferring bone marrow cells from WT (CD45.1) and Tbx21−/− (CD45.2) mice into CD45.1 lethally irradiated C57BL/6 host mice in a one to one ratio. We analyzed reconstitution in the spleen and found equivalent proportions of CD45.1+ and CD45.2+ lymphocytes, indicating that equal proportions of bone marrow precursors were transferred (). This ratio is maintained for total B (CD19+) and T (CD3ε+) cells. Consistent with our previous results, we found impaired generation of NKp46+ ILC22 and CD127+ NK cells in siLP and Peyer’s patches (PPs) in the absence of T-bet (CD45.2+ cells; ). Moreover, the residual NKp46+ ILC22 derived from Tbx21−/− bone marrow showed lower Rorγt expression and impaired ability to produce IL-22 (unpublished data). However, in this competitive setting we also noted reductions in LTi4 cells and even conventional NK cells present in the spleen (). Although lamina propria CD127− NK cells were not reduced (), T-bet deficiency was associated with defective IFN-γ production in these cells (). Thus, generation and/or function of diverse NKp46+ cells is influenced by T-bet.
Eomes is another T-box transcription factor, which can drive canonical NK cell generation in absence of T-bet (Gordon et al., 2012
). Consistent with the preservation of lamina propria CD127−
NK cells in mixed chimeras, these cells selectively express high levels of Eomes. Other ILC populations express little or no Eomes, providing an explanation for their dependency upon T-bet ().
ILC are developmentally related immune cells that play an important role in the regulation of gut homeostasis and protection from pathogens (Pearson et al., 2012
). The increasing complexity of ILC subsets to selectively produce cytokines beg the question as to what transcription factors are responsible for the generation of this diversity and how these subsets may or may not be related.
The present findings indicate that the establishment of NKp46+ ILC22 in the small intestine is tightly regulated by T-bet. The requirement of this transcription factor discriminates development of NKp46+ ILC22 from other ILC22 populations. In the residual NKp46+ ILC22 present in Tbx21−/− mice, both Rorγt expression and IL-22 production are decreased. These findings clearly warrant further exploration into the relationship of T-bet and IL-22 production. Based on the current findings, we favor the view that T-bet’s role in the regulation of IL-22 expression is likely related to impairment in differentiation. Although an alternative possibility is that it directly regulates IL-22 production, the production of this cytokine is not affected in other cells in the absence of T-bet. Ideally, one would want to tackle the problem of identifying T-bet targets in this cells using Chip-seq technology; however, the small numbers of these different cells preclude this approach at the present time.
Despite the dramatic effect of T-bet deficiency on NKp46+
, its function is not restricted to this subset. Even though LTi4
, like other Rorγt+
cells, is found in Tbx21−/−
mice, in a competitive environment the importance of T-bet becomes apparent. This observation indicates a wider role of T-bet in the generation and survival of various ILC subsets. In support of our present observation, Tbx21−/−Rag2−/−
mice develop colitis consistent with important actions on cells critical for gut homeostasis (Garrett et al., 2007
). Differentiated LTi cells express low levels of T-bet, so it is tempting to speculate that T-bet might regulate the turnover of a common ILC22 precursor. It would be of interest in the future to assess this possibility using a fate-mapping approach.
T-bet has previously been reported to control NK cell turnover and the trafficking of terminally differentiated NK cells (Townsend et al., 2004
; Jenne et al., 2009
). In addition, Trail+
NK cells present in the liver are maintained in a T-bet–dependent manner (Gordon et al., 2012
). Because the absence of T-bet also impairs generation of CD127+
NK cells, there may be a common progenitor for liver NK cells, gut CD127+
NK cells, and NKp46+
Like T-bet, Eomes is also a T-box family transcription factor. To some extent, its actions complement those of T-bet, driving NK cell generation when T-bet is not present (Gordon et al., 2012
). In intestinal ILC populations, we found high Eomes expression only in CD127−
NK cells, arguing for similarities with splenic NK cells. The high levels of Eomes expression may explain why these cells are resistant to loss of T-bet. Less obvious is why the absence of T-bet was associated with reduction of conventional splenic NK cells in a competitive bone marrow transplant and sparing of gut CD127−
NK cells. It is possible that this is the consequence of effects on trafficking versus survival. Regardless, the data argue for critical roles of T-box family transcription factors in the development and function of the ILCs.
Finally, our findings reveal added complexity in the developmental pathways leading to the generation of the various ILC populations. Because NKp46+ ILC22 depend on IL-7, AHR, and Rorγt and produce IL-22, it might be argued that their development is dependent on the same factors as LTi cells. In contrast, cells that are dependent on IL-15 include conventional NK cells (CD127−) in the bone marrow and spleen, and nonconventional NK cells, found in the liver (Trail+), thymus (CD127+), and gut (CD127+/−). However, this simple dichotomous model is probably not accurate. Rather, the data presented herein along with recently published results point to a more intricate and complex developmental model. For these cells, it appears that a network of transcription factors can shape their differentiation, such that the combination of T-bet, Eomes, and Rorγt expression could determine the distinct ILC subsets. The understanding of the extrinsic and intrinsic signals responsible for inducing the differential expression of these transcription factors remains an important but unresolved issue. Elucidating how these different transcription factors can influence the specification of different ILC will be an exciting future challenge.