The Multifaceted Roles of Bcl11b in Thymic and Peripheral T Cells: Impact on Immune Diseases

Bcl11b was initially discovered in neurons as CTIP2, along with its family member CTIP1 or Bcl11a (1), and it was later demonstrated to be critical for the proper development and function of neurons (2–5). Bcl11b is a C₂H₂ zinc finger protein that binds GC-rich response elements (6), and it was described to associate with a variety of cofactors, including the corepressor complexes NuRD (7, 8) and Sirt1 (9), as well as with the histone acetyltransferase (HAT) p300 (10), functioning both as a transcriptional repressor and activator (1, 6–13).

Bcl11b expression is initiated in a Notch1- and TCF-1–dependent manner at the double-negative (DN)2 stage of T cell development (14–17) and is maintained in mature T lymphocytes, including invariant NKT (iNKT) cells and regulatory T (Treg) cells (16, 18–23). It is expressed at lower levels in NK cells, but not in B cells, myeloid cells, or dendritic cells (16, 18, 20). In addition, Bcl11b was identified as a radiation-induced tumor suppressor gene, Rit1, in p53⁺ thymic lymphomas (24). Years of in-depth study revealed that Bcl11b is necessary for several developmental checkpoints, including T cell commitment at the DN2 stage (16, 25, 26), survival of DN3 and double-positive (DP) thymocytes (18, 27), β selection at the DN3 stage, positive selection of CD4 and CD8 single-positive (SP) thymocytes (18, 27, 28), development of Treg cells (22) and iNKT cells (23), and, most recently, development of a subpopulation of γδ T cells (29). In mature T cells, Bcl11b was demonstrated to control expansion and effector function of CTLs (19), restrict plasticity of Th17 cells by blocking expression of the Th2 program (21), control generation of inducible Treg (iTreg) cells from conventional CD4⁺ T cells, and, overall, to control the suppression function of Treg cells (22).

Dissecting the roles of Bcl11b in different cellular contexts and the factors that regulate Bcl11b provides valuable information for the growing transcriptional networks that involve Bcl11b, as well as for its implications in immune diseases. Furthermore, as new populations of immune cells are identified, such as innate lymphoid cells, novel Bcl11b targets and functions may be discovered.

The development of T cells is a multi-step process that requires environmental cues, including Notch signaling and cytokines, expression of key transcription factors necessary for T cell commitment, and downregulation of factors that support multi-potency and alternative lineages. Ellen Rothenberg’s group (14, 17, 25) showed that Bcl11b expression is initiated at the DN2a stage and continues to increase, reaching a peak at the DN2b stage, when self-renewal and multi-potency are suppressed. Using the OP-DL1 system, they demonstrated ex vivo that the absence of Bcl11b caused accumulation of DN1- and DN2a-stage thymocytes that expressed myeloid and NK lineage genes and failed to shut off stem cell and multi-potency genes (25) (Fig. 1). At the same time, Pentao Liu’s group (16) demonstrated that the loss of Bcl11b ex vivo caused reprogramming of DN2, DN3, and DP thymocytes to NK-like cells, designated induced T-to-NK (ITNK), which upregulated NK lineage genes, such as Id2, NK1.1, and NKp46, and possessed increased antitumor activity (see below). In a parallel study, Hiroshi Kawamoto’s group (26), despite earlier reports of a developmental block at the DN3 stage in global Bcl11b^−/− mice (27) (see below), concluded that the developmental block is rather at the DN2 stage (26). Despite the developmental block, the absolute numbers of Bcl11b^−/− DN2 thymocytes were not increased, although they vigorously proliferated and differentiated to NK and myeloid cells ex vivo (26), similar to what was reported in the other two studies (16, 25). Thus, these three studies all point to a critical role for Bcl11b at the DN2 stage in controlling the expression of genes that support T cell commitment and suppress multi-potency and alternative lineage genes (Fig. 1). It remains to be established whether Bcl11b^−/− DN2 thymocytes generated in vivo remain blocked at DN2 stage or differentiate into NK and myeloid cells.

As thymocytes enter the DN3 stage, TCR rearrangements occur at the TCRβ locus. Ryo Kominami’s group (27) analyzed neonatal thymi of global Bcl11b^−/− mice, which die soon after birth as a result of neuronal defects, and showed that thymocyte development was blocked at the DN3 stage, concomitant with diminished cellularity and increased apoptosis (Fig. 1). Additionally, TCRβ mRNA and protein levels were reduced as a result of impaired Vβ to Dβ rearrangements (27), restricting formation of the pre-TCR and halting T cell development. Provision of a transgenic TCR only partially rescued the defect (30), suggesting additional alterations. It remains to be established whether NK receptors, as well as other innate receptors, are expressed in vivo on Bcl11b^−/− DN3 thymocytes, similar to what was observed on ex vivo–generated Bcl11b^−/− DN3 thymocytes (16), which may interfere with appropriate signaling required for selection. Although γδT cells were reported to develop in normal numbers in the absence of Bcl11b (26, 27), more recent studies demonstrated that IL-17–producing γδT cells were absent in these mice (29).

Because of the developmental block at the DN2–DN3 stages, CD4-Cre–mediated deletion of Bcl11b, in which the Cre recombinase expression accumulates at the DP stage, was used to elucidate the role of Bcl11b in DP thymocytes (18, 28). We showed that these mice had a significant reduction in CD4 and CD8 SP thymocytes and peripheral CD4⁺ and CD8⁺ T cells, concomitant with an increase in some immune cell populations, including NK and myeloid cells. The increase in NK and myeloid cell numbers was associated with elevated splenic and bone marrow hematopoiesis through a bystander mechanism mediated by TNF-α, without indication of increased expansion (18, 31) (see below). The elevation in other immune populations, including B cells and γδ T cells, could be attributed to increased hematopoiesis and/or homeostatic expansion (18, 31). The reduction in peripheral T cells was caused by defective positive selection, associated with impaired TCR signaling (18) (Fig. 1). Although TCRα was normally rearranged, Bcl11b^−/− DP thymocytes had increased spontaneous apoptosis, which occurred even in the absence of TCR signaling, on a TCRα^−/− background (18). A slight decrease in the Bcl2 family member BCLXL and an increase in proapoptotic factors was observed; however, provision of the prosurvival factor Bcl2 only partially rescued survival, suggesting that additional factors are implicated (see below). Interestingly, neither provision of transgenic TCRs or Bcl2 rescued defective positive selection of Bcl11b^−/− DP thymocytes (18). Although reduced survival of DP thymocytes in the absence of Bcl11b is a common theme with DN3 thymocytes from global Bcl11b^−/− mice, Bcl11b^−/− DN2 thymocytes did not suffer from this defect, possibly related to their ex vivo generation on OP-DL1.

A large number of genes were found dysregulated in preselected Bcl11b^−/− DP thymocytes (18, 28) (GSE56714-mRNA from Bcl11b^−/− versus WT preselected DP thymocytes, D.I. Albu and Dr. Avram, unpublished observations). Some of these genes are known to play a role in TCR signaling (18). Additionally, genes with a role in commitment to the CD4 and CD8 lineages, such as Th-POK and Runx3 (32–34), were upregulated (28) (Fig. 1). Although Th-POK mRNA was one of the most upregulated in our microarrays as well, the protein could not be detected in Bcl11b^−/− DP thymocytes (GSE56714-mRNA from Bcl11b^−/− versus WT preselected DP thymocytes, D.I. Albu and D. Avram, unpublished observations), making it unlikely that its premature expression is the cause of altered positive selection and spontaneous apoptosis. mRNA for Id2, important for the NK program (35), also was upregulated (28), similar to Bcl11b^−/− DN2 thymocytes (16, 25), together with the mRNAs for the NK receptors Nkp46, Nk1.1, and CD244. Additionally, the mRNAs for several killer cell lectin-like receptors, including Klrd1, FcRs, Ly6C, and the inhibitory receptors PD-1 and CD160 (18, 28) were upregulated (and GSE56714-mRNA from Bcl11b^−/− versus WT preselected DP thymocytes, D.I. Albu and D. Avram, unpublished observations). Surprisingly, Bcl11b^−/− DP thymocytes did not present surface Nk1.1, Nkp46, or CD244 proteins (M. Uddin and D. Avram, unpublished observations), but they had elevated levels of surface CD160, PD-1, and Ly6C (18). We hypothesized that, in addition to the deregulated expression of some TCR-signaling components, the presence of Ly6C, CD160, and PD-1 inhibitory receptors on the surface of DP thymocytes may perturb TCR signaling and cause impaired positive selection. Interestingly, we also found alterations in several genes implicated in sphingolipid metabolism, which caused accumulation of sphingolipids, gangliosides, and cholesterol, affecting glycolipid presentation by DP thymocytes to iNKT precursors (23) (see below). Such alterations, in addition to being associated with enlarged lysosomes in Bcl11b^−/− DP thymocytes (23), can potentially affect membrane composition and consequently impair TCR signaling and selection, as well as survival (Fig. 1). Interestingly, Indra’s group (36) found later that Bcl11b^−/− embryonic skin had larger amounts of sphingomyelin. This raises the possibility that common pathways may be regulated by Bcl11b in skin, T cells, and, potentially, neurons.

Thus, Bcl11b has some common roles in DP, DN3, and DN2 thymocytes, such as repression of innate genes, and it supports the survival of both DP and DN3 thymocytes. However, Bcl11b also plays several distinctive functions in DP thymocytes: maintenance of expression of several components of TCR signaling, control of the expression of genes with critical roles in sphingolipid metabolism and cholesterol, and silencing of genes implicated in CD4 and CD8 T cell commitment, such as Th-POK and Runx3 (Fig. 1).

iNKT cells are a subset of T cells that express semi-invariant TCRs, composed of Vα14-Jα18 chains complexed with Vβ-chains of limited diversity. They are formed from DP thymocytes, which are also the cells that present self-glycolipids on CD1d molecules to select the iNKT precursors (37–39). Kastner et al. (28) showed that mice deficient for Bcl11b, starting with DP thymocytes, lack iNKT cells in the thymus and periphery. In a series of mixed bone marrow chimeras, which functionally separated DP thymocytes that present glycolipid from iNKT precursors, our group demonstrated that Bcl11b regulates iNKT development by both intrinsic and extrinsic mechanisms, specifically playing a dual role in the iNKT precursors and in the DP thymocytes that present glycolipids (23) (Fig. 1). The defect in glycolipid presentation was due to impaired sphingolipid metabolism, glycolipid trafficking, and loading on CD1d, with deregulated expression of cathepsin D and L genes, NPC1, and NPC2, as well as genes encoding lysosomal enzymes, such as β-galactosidase-1 and the acid sphingomyelinase (23). Sphingolipid species, including lactosylceramide, galactosylceramide, glucosylceramide, and sphingomyelin, as well as cholesterol, accumulated in Bcl11b^−/− DP thymocytes, which had enlarged lysosomes that were reminiscent of lysosomal storage disease (23).

The mechanisms by which iNKT cell development is intrinsically regulated by Bcl11b await clarification and are under investigation.

Thymic-derived or natural Treg (nTreg) cells also develop from DP thymocytes. iTreg cells are formed from conventional CD4⁺ T cells in the periphery in response to TGF-β and TCR stimulation (40, 41). Foxp3 is a critical transcription factor in the development of both nTreg cells and iTreg cells, and it acts in concert with other transcription factors and cofactors (42–44). Mutations at the Foxp3 locus cause dysregulation of Treg cell development and function, leading to lymphoproliferative diseases, fulminant autoimmunity, and death (45, 46). We found that Bcl11b^F/FCD4-Cre mice developed inflammatory bowel disease (IBD), which was rescued by the transfer of wild-type Treg cells, supporting the idea that inadequacy of Bcl11b^−/− Treg cells plays a critical role in IBD development in these mice (22). Furthermore, Bcl11b^F/FFoxp3-Cre mice had a similar phenotype, albeit delayed, which likely was associated with reduced levels of Cre in the Foxp3-Cre strain (47). Bcl11b^−/− Treg cells had reduced suppressive activity, reduced Foxp3 and IL-10 levels (both at the protein and mRNA levels), and upregulated proinflammatory cytokines, including TNF-α, IFN-γ, and IL-17, both at the mRNA and protein levels, acquiring overall a proinflammatory effector CD4⁺ T cell phenotype (22) (Fig. 1). In addition, genes identified before as being derepressed in the absence of Bcl11b in DN2 and DP thymocytes, such as NK receptors and Id2, were upregulated in Bcl11b^−/− Treg cells as well; however, similar to Bcl11b^−/− DP thymocytes (M. Uddin and D. Avram, unpublished observations) and Bcl11b^−/− CD4⁺ T cells (31), surface NK1.1 protein remained low (22). It is possible that DP thymocytes, CD4⁺ T cells, and Treg cells have additional mechanisms to restrict the presence of surface NK1.1 protein and other NK receptors, even when mRNAs are expressed. Chromatin immunoprecipitation assays revealed that Bcl11b associated with the promoter, CNS1, and CNS2 at the Foxp3 locus, thereby playing a critical role in the control of Foxp3 expression in both nTreg cells and iTreg cells and explaining why Bcl11b^−/− Treg cells have reduced Foxp3 levels (22). CNS1 was shown to be required for induction of Foxp3 expression during generation of iTreg cells from conventional CD4⁺ T cells (48). Supporting our observation, induction of Foxp3 in response to TCR activation and TGF-β treatment was reduced in the absence of Bcl11b, further establishing a role for Bcl11b in the generation of iTreg cells (22) (Fig. 1). Bcl11b^−/− Treg cells had reduced mRNA levels for several TGF-β–signaling components, including Tgfbr2, Smurf1, and Tgif2, suggesting the possibility that Bcl11b controls TGF-β signaling to induce Foxp3 in iTreg cells. It was demonstrated that TGF-β and TCR stimulation induces trimethylation of H3K4 at the CNS1 region, thereby opening the chromatin and allowing access of transcription factors, such as Smad3 (49). Furthermore, TGF-β stimulus inhibits the activity of Dnmt1, reducing DNA methylation status at CNS1, which is reversed once the stimulation stops (50). It is possible that Bcl11b acts similarly to Smad3, occupying the CNS1 region only when chromatin is opened and not having access when stimulation is removed. In conventional CD4⁺ T cells, in which Bcl11b is also expressed, the DNA is highly methylated at CNS1, likely blocking the access of factors that can induce Foxp3 expression (40), including Smad3 and, potentially, Bcl11b. Bcl11b’s relationship with Foxp3 is even more complex, given that Bcl11b was found to be part of the Foxp3 complex in Treg cells (51). Interestingly, we found that Bcl11b also associated with CNS2 (22), which is known to be bound by Foxp3 in a manner that is dependent on Runx-Cbfb (48, 52, 53). It remains to be established how Bcl11b works in concert with these complexes. Additionally, we found that Bcl11b regulates IL-10 gene expression by association with the promoter and CNS+6.5 (22). Therefore, in addition to the common role of restricting the expression of innate genes, Bcl11b plays specific roles in Treg cells, controlling Foxp3 and IL-10 expression and, overall, the suppression function of Treg cells. Additionally, it represses the effector CD4⁺ T cell program, and controls the generation of iTreg cells from conventional CD4⁺ T cells (22).

In addition to T cell development, Bcl11b is an important component of T cell effector function and differentiation. We recently demonstrated that Bcl11b restricts Th2 lineage gene expression in Th17 cells during experimental autoimmune encephalomyelitis (EAE) by repressing Gata-3 expression through direct interaction with the proximal Gata-3 promoter (21) (Fig. 1). Bivalent histone modifications, namely trimethylation of H3K4 and H3K27 at the promoters of lineage-determining transcription factors, such as Gata-3 and Tbet, which poise for transcriptional activation or repression, respectively, suggest that alternative lineage programs are not completely blocked following Th cell differentiation (54–56). Although Th17 cells were demonstrated previously to be plastic toward Treg cells and Th1 cells (56), to our knowledge, our study was the first to demonstrate that Bcl11b restricts plasticity toward the Th2 lineage genetic program (21). Despite the expression of IL-4 and Gata-3 (including at the protein level) in Bcl11b^−/− Th17 CD4⁺ T cells of mice with EAE, a Th17-mediated disease, the Th17 lineage transcription factor RORγt (57, 58) and the cytokines IL-17 and GM-CSF remained normal (21). Importantly, treatment promoting a Th2 response, by immunization with MOG35-55/Alum and Pam3CSK4, of EAE wild-type mice still did not affect the Th17 cytokines, demonstrating for the first time, to our knowledge, that the Th17 program is permissive to the Th2 genes (21), which has major implications for Th17-mediated autoimmune diseases. The main consequence of the common expression of IL-4 and Th17 cytokines by Bcl11b^−/− Th17 CD4⁺ T cells and by Th17 cells of wild-type mice with EAE vaccinated under Th2 conditions, was on their migration, through an extrinsic mechanism (21) (see below). Interestingly, although Bcl11b repressed Gata-3 gene expression in Th17 cells (21), Bcl11b was not required to downregulate Gata-3 in developing thymocytes, where Gata-3 seems to be upstream of Bcl11b (59), suggesting that, in such context, Bcl11b either does not have access or is competed out from Gata-3 promoter. It is possible that signaling events initiated by Th17-promoting cytokines, such as IL-6, TGF-β, IL-1β, and IL-23 (60), which are not likely to act in the thymus, play a critical role in this process, favoring recruitment of Bcl11b to the Gata-3 promoter, along with the NuRD complex, to deacetylate the histones and silence Gata-3 gene expression.

It is unknown specifically how Bcl11b is regulated to restrict Th17 cell plasticity, and it remains to be determined whether Bcl11b is important in promoting or restricting the plasticity of other effector CD4⁺ T cell populations.

Bcl11b is indispensable for effector CD8⁺ T cells, as well. Using conditional knockout mice, which express the Cre recombinase postselection, through the Lck distal promoter, we studied the role of Bcl11b in mature T cells, both Th cells (see above), and CD8⁺ T cells. In the steady-state, mice lacking Bcl11b in mature CD8⁺ T cells exhibited a reduction in CD8 expression, and Bcl11b occupied the enhancers E8I, IV, and V at the CD8 locus (19). Regulation of CD8 expression occurred only in CD8⁺ T cells and not in DP thymocytes (18, 19). Although expressed in CD4⁺ T cells, Bcl11b did not control the repression of CD8 gene in these cells (18, 19, 21). It is possible that other transcription factors, such as Th-POK, which occupy similar CD8 enhancer regions and mediate CD8 gene repression in CD4⁺ T cells via recruitment of histone deacetylases (61), may block Bcl11b’s access to these regions. Interestingly, in the steady-state, there was also a decrease in the naive CD8⁺ T cell pool in the absence of Bcl11b, and Bcl11b^−/− CD8⁺ T cells acquired a memory-like phenotype (D. Avram, unpublished observations), pointing to another potential role for Bcl11b in these cells, in controlling the naive state. Similar to Bcl11b^−/− DP thymocytes, the small number of Bcl11b^−/− naive CD8⁺ T cells upregulated Id2, PD-1, and CD160, in addition to NK1.1 and other NK receptors’ mRNAs, but not Th-POK mRNA (and GSE56925-mRNA from Bcl11b^−/− versus WT naive CD8⁺ T cells, D. Avram, unpublished observations).

Bcl11b^−/− effector CTLs had reduced expansion during the immune response to Listeria monocytogenes and influenza and diminished killing activity associated with low levels of granzyme B and perforin (19). Similar to DP thymocytes, Bcl11b^−/− CTLs had reduced TCR activation, with reduced levels of CD69, Zap70 phosphorylation, and calcium flux, and diminished mRNA levels for several components of TCR signaling, including CD3, CD28, and Plcg1 (19) (and GSE56713-mRNA from Bcl11b^−/− versus WT CD8⁺ T cells of mice infected with L. monocytogenes, day 6, D. Avram, unpublished observations). Bcl11b^−/− CTLs from mice infected with L. monocytogenes upregulated mRNAs for NK receptors but not for Id2, PD-1, or CD160 (and GSE56713-mRNA from Bcl11b^−/− versus WT CD8⁺ T cells of mice infected with L. monocytogenes day 6, D. Avram, unpublished observations). The fact that CTLs, known to express Id2 (13, 62), failed to further increase Id2 mRNA levels in the absence of Bcl11b might be caused by Id2 maximal levels, although we cannot exclude other mechanisms, such as exclusion from locus and/or competition with other transcription factors/complexes. Again, all these data suggest common themes, as well as differing roles, depending on the cell type, and moreover, indicate that, even in the same T cell type (CD8⁺ T cell, in this case), depending on the activation/effector state of the cell (naive versus CTL), Bcl11b differentially controls the expression of specific genes in a context-dependent manner.

Interestingly, once Bcl11b is expressed at the DN2 stage of T cell development, it remains expressed in all T cells. Kastner et al. (28) found that Bcl11b represses the expression of Th-POK and Runx3 genes in DP thymocytes; however, Bcl11b remains expressed in SP thymocytes and mature T cells, which require either Th-POK or Runx3, suggesting that the repressive function of Bcl11b at these genes must be blocked as T cells mature, similar to what happens to Id2 in CTLs. In addition, although Bcl11b does not seem to have an impact on Gata-3 during development, it represses its expression in Th17 cells (21).

We demonstrated that Bcl11b associates with the chromatin remodeling/histone deacetylase–containing complex NuRD and with HAT p300 to mediate transcriptional repression or activation, respectively (7, 10). Zhang et al. (13) recently demonstrated in thymocytes that the ability of Bcl11b to recruit the NuRD complex or p300 largely depends on its posttranslational modifications. Specifically, they showed that, in steady-state thymocytes, Bcl11b exists in several states—sumoylated, phosphorylated, and neither sumoylated nor phosphorylated, which all associate with the NuRD complex and repress Id2 gene expression. Stimulation of thymocytes with phorbol 12,13-dibutyrate and a calcium ionophore causes rapid and high phosphorylation of all three forms through the MAPK pathway, followed by desumoylation by SENP proteins. However, phosphatases are rapidly activated and dephosphorylate Bcl11b, which causes dismissal of SENP proteins and again allows sumoylation of Bcl11b, which now recruits the HAT p300 to derepress Id2, without replacement of NuRD (13). It remains to be established whether this mechanism occurs in the positive selection of DP thymocytes and whether Bcl11b functions as a transcriptional activator for the Id2 gene, in addition to being a transcriptional repressor, because its absence so far in early and DP thymocytes, as well as in naive CD8⁺ T cells, resulted in increased Id2 mRNA and no change in CTLs, but never downregulation in any tested Bcl11b^−/− T cell population or progenitor (16, 28) (GSE56714-mRNA from Bcl11b^−/− versus WT preselected DP thymocytes, GSE56925-mRNA from Bcl11b^−/− versus WT naive CD8⁺ T cells, and GSE56713-mRNA from Bcl11b GSE56714-mRNA from Bcl11b^−/− versus WT preselected DP thymocytes, GSE56925-mRNA from Bcl11b^−/− versus WT CD8⁺ T cells of mice infected with L. monocytogenes, day 6, D.I. Albu and D. Avram, unpublished observations). It also would be important to determine whether phosphorylation, sumoylation, and other posttranscriptional modifications modulate Bcl11b in other immune populations and at other loci (e.g., Th-pok, Runx3, Gata-3). The pattern that we observed in terms of regulation of a specific Bcl11b target is transcriptional repression or transcriptional activation in a given immune population and lack of regulation in another immune population, such as in the case of Id2, Th-pok, Runx3, and Gata-3. Additionally, we recently found that certain genes can be negatively regulated in one immune population and positively regulated in other immune populations; however, the two immune populations do not derive from each other (D. Califano and D. Avram, unpublished observations; M. Uddin and D. Avram, unpublished observations). We postulate that cell-specific signaling events trigger differential functions of Bcl11b, dependent on the cellular context. Such signaling events can be initiated by Notch in progenitors, TCR at selection stages, as well as TCR and/or cytokines during immune response, and further TCR and TGF-β in the conversion of conventional CD4⁺ T cells to iTreg cells.

The above-discussed data demonstrate the importance of Bcl11b in regulating a plethora of cellular functions in a cell-autonomous manner. In addition, our most recent data found that the absence of Bc11b in T cells can extrinsically alter the function and development of bystander cells and, importantly, greatly alter disease outcomes. The first evidence came from the role of Bcl11b in iNKT cell development, as described above, where Bcl11b deficiency in DP thymocytes resulted in a loss of iNKT selection due to impaired sphingolipid metabolism and glycolipid presentation to iNKT precursors (23). Two more recent studies also described extrinsically regulated defects in mice deficient in Bcl11b in specific T cell populations (21, 31). As discussed above, Bcl11b removal during Th17-induced EAE with the Lck distal promoter-Cre system, which acts only in mature T cells, enhanced Th17 cell plasticity by derepression of Gata-3 expression and IL-4 cytokine production, without restricting expression of the Th17 lineage transcription factor RORγt and production of IL-17 and GM-CSF (21). In this study, we demonstrated that coproduction of IL-4 and GM-CSF made dendritic cells, which do not express Bcl11b, upregulate RALDH2 expression, a key enzyme for production of retinoic acid (RA), and subsequently enhanced RA production (21). Our study further demonstrated that, during EAE, Bcl11b^−/− CD4⁺ T cells upregulated the gut-homing receptors integrin α4β7 and CCR9 through an extrinsic mechanism, which was dependent on RA and IL-4, causing their diversion from the draining lymph nodes/CNS to the mesenteric lymph nodes/small intestine. Furthermore, the diversion of Bcl11b^−/− effector CD4⁺ T cells caused a major reduction in EAE severity and, importantly, their presence in the small intestine did not result in symptoms of IBD. Bcl11b^−/− effector CD4⁺ T cells rerouted to the gut expressed suppressive cytokines without induction of Foxp3 expression (D. Califano and D. Avram, unpublished observations). Moreover, rerouting of the cells was recapitulated in wild-type mice with EAE that were treated with MOG_35–55 peptide under Th2-inducing conditions, bypassing the need for Bcl11b deficiency (21). These results demonstrate that induction of IL-4 is possible during Th17-mediated EAE, and it does not impact the Th17 cell program in itself; however, the combination of the two types of cytokines impacts trafficking of immune cells, which has a major impact on the disease outcome (21) and opens a novel therapeutic avenue for multiple sclerosis and other Th17-mediated autoimmune diseases.

As shown above, Pentao Liu’s group provided exciting evidence that the deletion of Bcl11b at early stages of thymic development or in DP thymocytes, with the use of an inducible ER-Cre system, reprogrammed the T cells to NK-like cells. In their experiments, Bcl11b^−/− DP thymocytes were generated ex vivo by treatment with tamoxifen for 48 h and then transferred in Rag2^−/−gc^−/− knockout mice, in which they formed mostly CD8⁺ T cells with enhanced antitumor activity (16). When we removed Bcl11b in vivo with the CD4-Cre system, the mice also exhibited reduced tumor burden in several tumor models, however in a manner independent of CD8⁺ T cells, but dependent on NK cells, CD4⁺ T cells and TNF-α, abundantly produced by Bcl11b^−/− CD4⁺ T cells (31). In our study, Bcl11b^−/− CD8⁺ T cells, although upregulated NK activating receptors, had minimal granzyme B levels and low degranulation, suggesting reduced effector function. Importantly, NK cell numbers were increased in Bcl11b^F/F/CD4-Cre mice and the only ones having high granzyme B levels and elevated degranulation (31). The increased numbers of NK cells resulted from elevated hematopoiesis in the bone marrow and spleen in a TNF-α–dependent manner (31). Bcl11b is not expressed in hematopoietic stem cells, but it is expressed in NK cells; however, the CD4-Cre system does not allow for its removal from NK cells, supporting the implication of another extrinsically regulated effect following Bcl11b removal through TNF-α. Importantly, low doses of TNF-α recapitulated stimulation of extracellular hematopoiesis and increased NK cell formation, as well as increased the antitumor immune response (31). The observed differences between our study and that by Liu’s group may be due to the fact that the initiation of ITNK cell generation ex vivo in their study is more efficient, lacking the restrictions imposed by thymic selection, which likely would eliminate highly reactive cells. In our study with Bcl11b^F/F/CD4-Cre mice, the small number of Bcl11b^−/− CD8⁺ T cells formed is likely to be strictly controlled by other immune populations and exposed to a different environment than are the ITNK cells transferred in Rag2^−/−gc^−/− mice, including the fact they do not have the opportunity to expand as vigorously as in Rag2^−/−gc^−/− recipient mice.

One common theme when Bcl11b is removed in T cells is the expression of innate cell genes, supporting the idea that, overall, Bcl11b represses such a program in T cells. Another theme in several progenitors and mature CD8⁺ T cells is the expression maintenance of genes implicated in TCR signaling. Remarkably, an important function of Bcl11b in DP thymocytes and developing skin is the control of sphingolipid metabolism; it remains to be determined whether it is a common theme in other T cell progenitors and subsets, as well as in neurons. In addition to the common themes, we postulate that Bcl11b has diverse roles, controlling specific genes, depending on the T cell developmental stage and on the T cell subset and, moreover, having differing roles depending on the T cell activation state. Such diverse and complex roles support the idea that Bcl11b’s activity is dependent on stage- and subset-specific transcription factors and cofactors, as well as on cell type–specific signaling events. How Bcl11b is integrated in such complex regulatory networks remains to be further elucidated.

The authors have no financial conflicts of interest.