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

This information is current as Dorina Avram and Danielle Califano of September 29, 2021. J Immunol 2014; 193:2059-2065; ; doi: 10.4049/jimmunol.1400930 http://www.jimmunol.org/content/193/5/2059 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Th eJournal of Brief Reviews Immunology

The Multifaceted Roles of Bcl11b in Thymic and Peripheral T Cells: Impact on Immune Diseases Dorina Avram and Danielle Califano The factor Bcl11b is expressed in all T cell commitment at the DN2 stage (16, 25, 26), survival of subsets and progenitors, starting from the DN2 stage of DN3 and double-positive (DP) (18, 27), b se- T cell development, and it regulates critical processes lection at the DN3 stage, positive selection of CD4 and CD8 implicated in the development, function, and survival single-positive (SP) thymocytes (18, 27, 28), development of of many of these cells. Among the common roles of Treg cells (22) and iNKT cells (23), and, most recently, de- Bcl11b in T cell progenitors and mature T cell subsets velopment of a subpopulation of gd T cells (29). In mature are the repression of the innate genetic program and, to T cells, Bcl11b was demonstrated to control expansion and some extent, expression maintenance of TCR-signaling effector function of CTLs (19), restrict plasticity of Th17 cells Downloaded from components. However, Bcl11b also has unique roles in by blocking expression of the Th2 program (21), control generation of inducible Treg (iTreg) cells from conventional specific T cell populations, suggesting that its functions + depend on cell type and activation state of the cell. In this CD4 T cells, and, overall, to control the suppression func- article, we provide a comprehensive review of the roles of tion of Treg cells (22). Dissecting the roles of Bcl11b in different cellular contexts and Bcl11b in progenitors, effector T cells, regulatory T cells, the factors that regulate Bcl11b provides valuable information for http://www.jimmunol.org/ and invariant NKT cells, as well as its impact on im- the growing transcriptional networks that involve Bcl11b, as well mune diseases. While emphasizing common themes, as for its implications in immune diseases. Furthermore, as new including some that might be extended to skin and neu- populations of immune cells are identified, such as innate lym- rons, we also describe the control of specific functions phoid cells, novel Bcl11b targets and functions may be discovered. in different T cell subsets. The Journal of Immunology, 2014, 193: 2059–2065. Bcl11b is essential for multiple checkpoints during T cell development Bcl11b in early stages of T cell development: repression of multi-potency

cl11b was initially discovered in neurons as CTIP2, and alternate lineage potential and enforcement of commitment. The by guest on September 29, 2021 along with its family member CTIP1 or Bcl11a (1), development of T cells is a multi-step process that requires B and it was later demonstrated to be critical for the environmental cues, including Notch signaling and , proper development and function of neurons (2–5). Bcl11b is expression of key transcription factors necessary for T cell com- mitment, and downregulation of factors that support multi- aC2H2 zinc finger that binds GC-rich response elements (6), and it was described to associate with a variety of cofactors, potency and alternative lineages. Ellen Rothenberg’s group (14, 17, 25) showed that Bcl11b expression is initiated at the including the corepressor complexes NuRD (7, 8) and Sirt1 (9), as DN2a stage and continues to increase, reaching a peak at the well as with the histone acetyltransferase (HAT) p300 (10), func- DN2b stage, when self-renewal and multi-potency are sup- tioning both as a transcriptional repressor and activator (1, 6–13). pressed. Using the OP-DL1 system, they demonstrated ex vivo Bcl11b expression is initiated in a Notch1- and TCF-1– that the absence of Bcl11b caused accumulation of DN1- and dependent manner at the double-negative (DN)2 stage of DN2a-stage thymocytes that expressed myeloid and NK lineage T cell development (14–17) and is maintained in mature and failed to shut off stem cell and multi-potency genes T , including invariant NKT (iNKT) cells and (25) (Fig. 1). At the same time, Pentao Liu’s group (16) demon- regulatory T (Treg) cells (16, 18–23). It is expressed at lower strated that the loss of Bcl11b ex vivo caused reprogramming of levels in NK cells, but not in B cells, myeloid cells, or den- DN2, DN3, and DP thymocytes to NK-like cells, designated dritic cells (16, 18, 20). In addition, Bcl11b was identified as a induced T-to-NK (ITNK), which upregulated NK lineage radiation-induced tumor suppressor , Rit1,inp53+ thymic genes, such as Id2, NK1.1, and NKp46, and possessed increased (24). Years of in-depth study revealed that Bcl11b antitumor activity (see below). In a parallel study, Hiroshi is necessary for several developmental checkpoints, including Kawamoto’s group (26), despite earlier reports of a developmental

Center for Immunology and Microbial Disease, Albany Medical College, Albany, NY Abbreviations used in this article: DN, double negative; DP, double positive; EAE, 12208 experimental autoimmune encephalomyelitis; HAT, histone acetyltransferase; IBD, in- flammatory bowel disease; iNKT, invariant NKT; ITNK, induced T-to-NK; iTreg, in- Received for publication April 10, 2014. Accepted for publication June 25, 2014. ducible Treg; nTreg, thymic-derived or natural Treg; RA, retinoic acid; SP, single positive; This work was supported by National Institute of Allergy and Infectious Diseases, Treg, regulatory T. National institutes of Health Grants AI067846 and AI078273 (to D.A.). Ó Address correspondence and reprint requests to Dr. Dorina Avram, Center for Immu- Copyright 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 nology and Microbial Disease, Albany Medical College, 47 New Scotland Avenue, Albany, NY 12208. E-mail address: [email protected]

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1400930 2060 BRIEF REVIEWS: Bcl11b IN T CELLS AND IMMUNE DISEASES

2 2 block at the DN3 stage in global Bcl11b / mice (27) (see below), of Bcl11b, in which the Cre recombinase expression accu- concluded that the developmental block is rather at the DN2 stage mulates at the DP stage, was used to elucidate the role of (26). Despite the developmental block, the absolute numbers of Bcl11b in DP thymocytes (18, 28). We showed that these 2 2 Bcl11b / DN2 thymocytes were not increased, although they mice had a significant reduction in CD4 and CD8 SP thy- vigorously proliferated and differentiated to NK and myeloid cells mocytes and peripheral CD4+ and CD8+ T cells, concomitant ex vivo (26), similar to what was reported in the other two studies with an increase in some immune cell populations, including NK (16, 25). Thus, these three studies all point to a critical role for and myeloid cells. The increase in NK and myeloid cell numbers Bcl11b at the DN2 stage in controlling the expression of genes was associated with elevated splenic and bone marrow that support T cell commitment and suppress multi-potency and hematopoiesis through a bystander mechanism mediated by alternative lineage genes (Fig. 1). It remains to be established TNF-a, without indication of increased expansion (18, 31) 2/2 whether Bcl11b DN2 thymocytes generated in vivo remain (see below). The elevation in other immune populations, blocked at DN2 stage or differentiate into NK and myeloid cells. including B cells and gd T cells, could be attributed to in- Bcl11b at DN3 stage and b selection. As thymocytes enter the creased hematopoiesis and/or homeostatic expansion (18, 31). DN3 stage, TCR rearrangements occur at the TCRb . The reduction in peripheral T cells was caused by defective Ryo Kominami’s group (27) analyzed neonatal thymi of positive selection, associated with impaired TCR signaling (18) 2 2 2 2 global Bcl11b / mice, which die soon after birth as a result (Fig. 1). Although TCRa was normally rearranged, Bcl11b / of neuronal defects, and showed that development DP thymocytes had increased spontaneous apoptosis, which 2 2 was blocked at the DN3 stage, concomitant with diminished occurredevenintheabsenceofTCRsignaling,onaTCRa / Downloaded from cellularity and increased apoptosis (Fig. 1). Additionally, background (18). A slight decrease in the Bcl2 family member TCRb mRNA and protein levels were reduced as a result of BCLXL and an increase in proapoptotic factors was observed; impaired Vb to Db rearrangements (27), restricting formation however, provision of the prosurvival factor Bcl2 only partially of the pre-TCR and halting T cell development. Provision rescued survival, suggesting that additional factors are implicated of a transgenic TCR only partially rescued the defect (30), (see below). Interestingly, neither provision of transgenic TCRs

2 2 http://www.jimmunol.org/ suggesting additional alterations. It remains to be established or Bcl2 rescued defective positive selection of Bcl11b / DP whether NK receptors, as well as other innate receptors, are thymocytes (18). Although reduced survival of DP thymocytes 2/2 expressed in vivo on Bcl11b DN3 thymocytes, similar in the absence of Bcl11b is a common theme with DN3 2/2 2 2 2 2 towhatwasobservedonexvivo–generatedBcl11b thymocytes from global Bcl11b / mice, Bcl11b / DN2 DN3 thymocytes (16), which may interfere with appropriate thymocytes did not suffer from this defect, possibly related to signaling required for selection. Although gdT cells were their ex vivo generation on OP-DL1. reported to develop in normal numbers in the absence of A large number of genes were found dysregulated in pre- 2 2 Bcl11b (26, 27), more recent studies demonstrated that IL- selected Bcl11b / DP thymocytes (18, 28) (GSE56714- 2/2 17–producing gdT cells were absent in these mice (29). mRNA from Bcl11b versus WT preselected DP thymo- by guest on September 29, 2021 Bcl11b in DP thymocytes: role in positive selection, survival, cytes, D.I. Albu and Dr. Avram, unpublished observations). sphingolipid metabolism, and repression of genes involved in CD4 Some of these genes are known to play a role in TCR signaling and CD8 lineage commitment. Because of the developmental (18). Additionally, genes with a role in commitment to the block at the DN2–DN3 stages, CD4-Cre–mediated deletion CD4 and CD8 lineages, such as Th-POK and Runx3 (32–34),

FIGURE 1. Roles of Bcl11b in thymic and peripheral T cells. Thymic developmental stages are indicated as double negative (DN1–DN4), DP, or SP. Specific T cell populations are indicated as iNKT, nTreg, iTreg, CD4+,CD8+, Th17 T cells, and CTLs. T cell populations and processes in which Bcl11b plays a role are shown in red. Functions controlled by Bcl11b in each developmental stage or T cell subsets are indicated. The Journal of Immunology 2061 were upregulated (28) (Fig. 1). Although Th-POK mRNA was due to impaired sphingolipid metabolism, glycolipid was one of the most upregulated in our microarrays as well, trafficking, and loading on CD1d, with deregulated expres- 2 2 the protein could not be detected in Bcl11b / DP thymo- sion of cathepsin D and L genes, NPC1, and NPC2, as well as 2 2 cytes (GSE56714-mRNA from Bcl11b / versus WT pre- genes encoding lysosomal enzymes, such as b-galactosidase-1 selected DP thymocytes, D.I. Albu and D. Avram, un- and the acid sphingomyelinase (23). Sphingolipid species, published observations), making it unlikely that its premature including lactosylceramide, galactosylceramide, glucosylcer- expression is the cause of altered positive selection and amide, and sphingomyelin, as well as cholesterol, accumulated 2 2 spontaneous apoptosis. mRNA for Id2, important for the NK in Bcl11b / DP thymocytes, which had enlarged lysosomes 2 2 program (35), also was upregulated (28), similar to Bcl11b / that were reminiscent of lysosomal storage disease (23). DN2 thymocytes (16, 25), together with the mRNAs for the The mechanisms by which iNKT cell development is in- NK receptors Nkp46, Nk1.1, and CD244. Additionally, the trinsically regulated by Bcl11b await clarification and are under mRNAs for several killer cell lectin-like receptors, including investigation. Klrd1, FcRs, Ly6C, and the inhibitory receptors PD-1 and CD160 (18, 28) were upregulated (and GSE56714-mRNA Bcl11b in Treg cells 2/2 from Bcl11b versus WT preselected DP thymocytes, Thymic-derived or natural Treg (nTreg) cells also develop D.I. Albu and D. Avram, unpublished observations). Sur- from DP thymocytes. iTreg cells are formed from conventional 2/2 prisingly, Bcl11b DP thymocytes did not present surface CD4+ T cells in the periphery in response to TGF-b and Nk1.1, Nkp46, or CD244 (M. Uddin and D. Avram, TCR stimulation (40, 41). Foxp3 is a critical transcription Downloaded from unpublished observations), but they had elevated levels of sur- factor in the development of both nTreg cells and iTreg cells, face CD160, PD-1, and Ly6C (18). We hypothesized that, in and it acts in concert with other transcription factors and addition to the deregulated expression of some TCR-signaling cofactors (42–44). at the Foxp3 locus cause dys- components, the presence of Ly6C, CD160, and PD-1 in- regulation of Treg cell development and function, leading to hibitory receptors on the surface of DP thymocytes may perturb lymphoproliferative diseases, fulminant , and TCR signaling and cause impaired positive selection. Interest- death (45, 46). We found that Bcl11bF/FCD4-Cre mice de- http://www.jimmunol.org/ ingly, we also found alterations in several genes implicated veloped inflammatory bowel disease (IBD), which was res- in sphingolipid metabolism, which caused accumulation of cued by the transfer of wild-type Treg cells, supporting the 2 2 sphingolipids, gangliosides, and cholesterol, affecting glycolipid idea that inadequacy of Bcl11b / Treg cells plays a critical presentation by DP thymocytes to iNKT precursors (23) (see role in IBD development in these mice (22). Furthermore, below). Such alterations, in addition to being associated with Bcl11bF/FFoxp3-Cre mice had a similar , albeit 2/2 enlarged lysosomes in Bcl11b DP thymocytes (23), can delayed, which likely was associated with reduced levels of Cre 2 2 potentially affect membrane composition and consequently in the Foxp3-Cre strain (47). Bcl11b / Treg cells had re- impair TCR signaling and selection, as well as survival (Fig. 1). duced suppressive activity, reduced Foxp3 and IL-10 levels by guest on September 29, 2021 2/2 Interestingly, Indra’s group (36) found later that Bcl11b (both at the protein and mRNA levels), and upregulated embryonic skin had larger amounts of sphingomyelin. This proinflammatory cytokines, including TNF-a, IFN-g,and raises the possibility that common pathways may be regulated IL-17, both at the mRNA and protein levels, acquiring over- by Bcl11b in skin, T cells, and, potentially, neurons. all a proinflammatory effector CD4+ T cell phenotype (22) Thus, Bcl11b has some common roles in DP, DN3, and (Fig. 1). In addition, genes identified before as being dere- DN2 thymocytes, such as repression of innate genes, and it pressed in the absence of Bcl11b in DN2 and DP thymocytes, 2 2 supports the survival of both DP and DN3 thymocytes. such as NK receptors and Id2, were upregulated in Bcl11b / 2 2 However, Bcl11b also plays several distinctive functions in DP Treg cells as well; however, similar to Bcl11b / DP thy- thymocytes: maintenance of expression of several components mocytes (M. Uddin and D. Avram, unpublished observa- 2 2 of TCR signaling, control of the expression of genes with tions) and Bcl11b / CD4+ T cells (31), surface NK1.1 critical roles in sphingolipid metabolism and cholesterol, and protein remained low (22). It is possible that DP thymocytes, silencing of genes implicated in CD4 and CD8 T cell com- CD4+ T cells, and Treg cells have additional mechanisms to mitment, such as Th-POK and Runx3 (Fig. 1). restrict the presence of surface NK1.1 protein and other NK receptors, even when mRNAs are expressed. Chromatin im- Bcl11b in iNKT cells: intrinsic and extrinsic roles munoprecipitation assays revealed that Bcl11b associated iNKT cells are a subset of T cells that express semi-invariant with the , CNS1, and CNS2 at the Foxp3 locus, TCRs, composed of Va14-Ja18 chains complexed with Vb- thereby playing a critical role in the control of Foxp3 ex- chains of limited diversity. They are formed from DP thy- pression in both nTreg cells and iTreg cells and explaining 2 2 mocytes, which are also the cells that present self-glycolipids why Bcl11b / Treg cells have reduced Foxp3 levels (22). on CD1d molecules to select the iNKT precursors (37–39). CNS1 was shown to be required for induction of Foxp3 ex- Kastner et al. (28) showed that mice deficient for Bcl11b, pression during generation of iTreg cells from conventional starting with DP thymocytes, lack iNKT cells in the CD4+ T cells (48). Supporting our observation, induction of and periphery. In a series of mixed bone marrow chimeras, Foxp3 in response to TCR activation and TGF-b treatment which functionally separated DP thymocytes that present was reduced in the absence of Bcl11b, further establishing glycolipid from iNKT precursors, our group demonstrated a role for Bcl11b in the generation of iTreg cells (22) (Fig. 1). 2 2 that Bcl11b regulates iNKT development by both intrinsic Bcl11b / Treg cells had reduced mRNA levels for several and extrinsic mechanisms, specifically playing a dual role in TGF-b–signaling components, including Tgfbr2, Smurf1, and the iNKT precursors and in the DP thymocytes that present Tgif2, suggesting the possibility that Bcl11b controls TGF-b glycolipids (23) (Fig. 1). The defect in glycolipid presentation signaling to induce Foxp3 in iTreg cells. It was demonstrated 2062 BRIEF REVIEWS: Bcl11b IN T CELLS AND IMMUNE DISEASES that TGF-b and TCR stimulation induces trimethylation of regulate Gata-3 in developing thymocytes, where Gata-3 H3K4 at the CNS1 region, thereby opening the chromatin and seems to be upstream of Bcl11b (59), suggesting that, in such allowing access of transcriptionfactors,suchasSmad3(49). context, Bcl11b either does not have access or is competed out Furthermore, TGF-b stimulus inhibits the activity of Dnmt1, from Gata-3 promoter. It is possible that signaling events reducing DNA methylation statusatCNS1,whichisreversed initiated by Th17-promoting cytokines, such as IL-6, TGF-b, once the stimulation stops (50). It is possible that Bcl11b acts IL-1b, and IL-23 (60), which are not likely to act in the similarly to Smad3, occupying the CNS1 region only when thymus, play a critical role in this process, favoring recruit- chromatin is opened and not having access when stimulation ment of Bcl11b to the Gata-3 promoter, along with the is removed. In conventional CD4+ T cells, in which Bcl11b is NuRD complex, to deacetylate the histones and silence Gata-3 also expressed, the DNA is highly methylated at CNS1, likely . blocking the access of factors that can induce Foxp3 expression It is unknown specifically how Bcl11b is regulated to restrict (40), including Smad3 and, potentially, Bcl11b. Bcl11b’s - Th17 cell plasticity, and it remains to be determined whether tionship with Foxp3 is even more complex, given that Bcl11b Bcl11b is important in promoting or restricting the plasticity was found to be part of the Foxp3 complex in Treg cells (51). of other effector CD4+ T cell populations. Interestingly, we found that Bcl11b also associated with CNS2 + (22), which is known to be bound by Foxp3 in a manner that is Bcl11b in mature CD8 T cells dependent on Runx-Cbfb (48, 52, 53). It remains to be estab- Bcl11b is indispensable for effector CD8+ T cells, as well. lished how Bcl11b works in concert with these complexes. Ad- Using conditional knockout mice, which express the Cre Downloaded from ditionally, we found that Bcl11b regulates IL-10 gene expression recombinase postselection, through the Lck distal promoter, by association with the promoter and CNS+6.5 (22). Therefore, we studied the role of Bcl11b in mature T cells, both Th cells in addition to the common role of restricting the expression of (see above), and CD8+ T cells. In the steady-state, mice innate genes, Bcl11b plays specific roles in Treg cells, controlling lacking Bcl11b in mature CD8+ T cells exhibited a reduction Foxp3 and IL-10 expression and, overall, the suppression func- in CD8 expression, and Bcl11b occupied the enhancers E8I, + tion of Treg cells. Additionally, it represses the effector CD4 IV, and V at the CD8 locus (19). Regulation of CD8 ex- http://www.jimmunol.org/ T cell program, and controls the generation of iTreg cells from pression occurred only in CD8+ T cells and not in DP thy- + conventional CD4 T cells (22). mocytes (18, 19). Although expressed in CD4+ T cells, Bcl11b did not control the repression of CD8 gene in these Bcl11b restricts Th17 cell plasticity by repressing the Th2 program cells (18, 19, 21). It is possible that other transcription factors, In addition to T cell development, Bcl11b is an important such as Th-POK, which occupy similar CD8 enhancer regions component of T cell effector function and differentiation. We and mediate CD8 gene repression in CD4+ T cells via re- recently demonstrated that Bcl11b restricts Th2 lineage gene cruitment of histone deacetylases (61), may block Bcl11b’s expression in Th17 cells during experimental autoimmune access to these regions. Interestingly, in the steady-state, there by guest on September 29, 2021 encephalomyelitis (EAE) by repressing Gata-3 expression was also a decrease in the naive CD8+ T cell pool in the 2 2 through direct interaction with the proximal Gata-3 promoter absence of Bcl11b, and Bcl11b / CD8+ T cells acquired (21) (Fig. 1). Bivalent histone modifications, namely trime- a memory-like phenotype (D. Avram, unpublished observa- thylation of H3K4 and H3K27 at the promoters of lineage- tions), pointing to another potential role for Bcl11b in these 2 2 determining transcription factors, such as Gata-3 and Tbet, cells, in controlling the naive state. Similar to Bcl11b / DP 2 2 which poise for transcriptional activation or repression, re- thymocytes, the small number of Bcl11b / naive CD8+ spectively, suggest that alternative lineage programs are not T cells upregulated Id2, PD-1, and CD160, in addition to completely blocked following Th cell differentiation (54–56). NK1.1 and other NK receptors’ mRNAs, but not Th-POK 2 2 Although Th17 cells were demonstrated previously to be mRNA (and GSE56925-mRNA from Bcl11b / versus WT plastic toward Treg cells and Th1 cells (56), to our knowl- naive CD8+ T cells, D. Avram, unpublished observations). 2 2 edge, our study was the first to demonstrate that Bcl11b Bcl11b / effector CTLs had reduced expansion during restricts plasticity toward the Th2 lineage genetic program the immune response to Listeria monocytogenes and influenza (21). Despite the expression of IL-4 and Gata-3 (including at and diminished killing activity associated with low levels of 2 2 the protein level) in Bcl11b / Th17 CD4+ T cells of mice granzyme B and perforin (19). Similar to DP thymocytes, 2 2 with EAE, a Th17-mediated disease, the Th17 lineage tran- Bcl11b / CTLs had reduced TCR activation, with reduced scription factor RORgt (57, 58) and the cytokines IL-17 and levels of CD69, Zap70 phosphorylation, and calcium flux, GM-CSF remained normal (21). Importantly, treatment and diminished mRNA levels for several components of promoting a Th2 response, by immunization with MOG35- TCR signaling, including CD3, CD28, and Plcg1 (19) (and 2 2 55/Alum and Pam3CSK4, of EAE wild-type mice still did not GSE56713-mRNA from Bcl11b / versus WT CD8+ T cells affect the Th17 cytokines, demonstrating for the first time, to of mice infected with L. monocytogenes, day 6, D. Avram, 2 2 our knowledge, that the Th17 program is permissive to the unpublished observations). Bcl11b / CTLs from mice Th2 genes (21), which has major implications for Th17- infected with L. monocytogenes upregulated mRNAs for NK mediated autoimmune diseases. The main consequence of receptors but not for Id2, PD-1, or CD160 (and GSE56713- 2 2 the common expression of IL-4 and Th17 cytokines by mRNA from Bcl11b / versus WT CD8+ T cells of mice 2 2 Bcl11b / Th17 CD4+ T cells and by Th17 cells of wild-type infected with L. monocytogenes day 6, D. Avram, unpublished mice with EAE vaccinated under Th2 conditions, was on their observations). The fact that CTLs, known to express Id2 (13, migration, through an extrinsic mechanism (21) (see below). 62), failed to further increase Id2 mRNA levels in the absence Interestingly, although Bcl11b repressed Gata-3 gene expres- of Bcl11b might be caused by Id2 maximal levels, although sion in Th17 cells (21), Bcl11b was not required to down- we cannot exclude other mechanisms, such as exclusion from The Journal of Immunology 2063 locus and/or competition with other transcription factors/ a given immune population and lack of regulation in another complexes. Again, all these data suggest common themes, as immune population, such as in the case of Id2, Th-pok, well as differing roles, depending on the cell type, and Runx3, and Gata-3. Additionally, we recently found that moreover, indicate that, even in the same T cell type (CD8+ certain genes can be negatively regulated in one immune T cell, in this case), depending on the activation/effector population and positively regulated in other immune pop- state of the cell (naive versus CTL), Bcl11b differentially ulations; however, the two immune populations do not derive controls the expression of specific genes in a context-dependent from each other (D. Califano and D. Avram, unpublished manner. observations;M.UddinandD.Avram,unpublishedobserva- tions). We postulate that cell-specific signaling events trigger Modulation of Bcl11b function is dependent on posttranslational differential functions of Bcl11b, dependent on the cellular modifications context. Such signaling events can be initiated by Notch in Interestingly, once Bcl11b is expressed at the DN2 stage of progenitors, TCR at selection stages, as well as TCR and/or T cell development, it remains expressed in all T cells. Kastner cytokines during immune response, and further TCR and TGF-b + et al. (28) found that Bcl11b represses the expression of Th- in the conversion of conventional CD4 T cells to iTreg cells. POK and Runx3 genes in DP thymocytes; however, Bcl11b remains expressed in SP thymocytes and mature T cells, Bcl11b extrinsically alters the function of several immune populations which require either Th-POK or Runx3, suggesting that the and disease outcomes repressive function of Bcl11b at these genes must be blocked Downloaded from The above-discussed data demonstrate the importance of as T cells mature, similar to what happens to Id2 in CTLs. In Bcl11b in regulating a plethora of cellular functions in a cell- addition, although Bcl11b does not seem to have an impact autonomous manner. In addition, our most recent data found on Gata-3 during development, it represses its expression in that the absence of Bc11b in T cells can extrinsically alter the Th17 cells (21). function and development of bystander cells and, importantly, We demonstrated that Bcl11b associates with the chromatin

greatly alter disease outcomes. The first evidence came from http://www.jimmunol.org/ remodeling/histone deacetylase–containing complex NuRD the role of Bcl11b in iNKT cell development, as described and with HAT p300 to mediate transcriptional repression or above, where Bcl11b deficiency in DP thymocytes resulted in activation, respectively (7, 10). Zhang et al. (13) recently a loss of iNKT selection due to impaired sphingolipid meta- demonstrated in thymocytes that the ability of Bcl11b to bolism and glycolipid presentation to iNKT precursors (23). recruit the NuRD complex or p300 largely depends on its Two more recent studies also described extrinsically regulated posttranslational modifications. Specifically, they showed that, in steady-state thymocytes, Bcl11b exists in several states— defects in mice deficient in Bcl11b in specific T cell popula- sumoylated, phosphorylated, and neither sumoylated nor tions (21, 31). As discussed above, Bcl11b removal during Th17-induced EAE with the Lck distal promoter-Cre system, phosphorylated, which all associate with the NuRD complex by guest on September 29, 2021 and repress Id2 gene expression. Stimulation of thymocytes which acts only in mature T cells, enhanced Th17 cell plasticity with phorbol 12,13-dibutyrate and a calcium ionophore by derepression of Gata-3 expression and IL-4 pro- causes rapid and high phosphorylation of all three forms duction, without restricting expression of the Th17 lineage through the MAPK pathway, followed by desumoylation by RORgt and production of IL-17 and GM- SENP proteins. However, phosphatases are rapidly activated CSF (21). In this study, we demonstrated that coproduction of and dephosphorylate Bcl11b, which causes dismissal of SENP IL-4 and GM-CSF made dendritic cells, which do not express proteins and again allows sumoylation of Bcl11b, which now Bcl11b, upregulate RALDH2 expression, a key enzyme for recruits the HAT p300 to derepress Id2, without replacement production of retinoic acid (RA), and subsequently enhanced RA production (21). Our study further demonstrated that, of NuRD (13). It remains to be established whether this 2/2 + mechanism occurs in the positive selection of DP thymocytes during EAE, Bcl11b CD4 T cells upregulated the gut- and whether Bcl11b functions as a transcriptional activator homing receptors integrin a4b7 and CCR9 through an ex- for the Id2 gene, in addition to being a transcriptional re- trinsic mechanism, which was dependent on RA and IL-4, pressor, because its absence so far in early and DP thymocytes, causing their diversion from the draining lymph nodes/CNS as well as in naive CD8+ T cells, resulted in increased Id2 to the mesenteric lymph nodes/small intestine. Furthermore, 2/2 + mRNA and no change in CTLs, but never downregulation in the diversion of Bcl11b effector CD4 T cells caused 2 2 any tested Bcl11b / T cell population or progenitor (16, 28) a major reduction in EAE severity and, importantly, their 2 2 (GSE56714-mRNA from Bcl11b / versus WT preselected presence in the small intestine did not result in symptoms of 2 2 2/2 + DP thymocytes, GSE56925-mRNA from Bcl11b / versus IBD. Bcl11b effector CD4 T cells rerouted to the gut WT naive CD8+ T cells, and GSE56713-mRNA from Bcl11b expressed suppressive cytokines without induction of Foxp3 2 2 GSE56714-mRNA from Bcl11b / versus WT preselected expression (D. Califano and D. Avram, unpublished ob- 2 2 DP thymocytes, GSE56925-mRNA from Bcl11b / versus servations). Moreover, rerouting of the cells was recapitulated + WT CD8 T cells of mice infected with L. monocytogenes, in wild-type mice with EAE that were treated with MOG35–55 day 6, D.I. Albu and D. Avram, unpublished observations). peptide under Th2-inducing conditions, bypassing the need It also would be important to determine whether phosphor- for Bcl11b deficiency (21). These results demonstrate that ylation, sumoylation, and other posttranscriptional mod- induction of IL-4 is possible during Th17-mediated EAE, and ifications modulate Bcl11b in other immune populations and it does not impact the Th17 cell program in itself; however, at other loci (e.g., Th-pok, Runx3, Gata-3). The pattern that the combination of the two types of cytokines impacts traf- we observed in terms of regulation of a specific Bcl11b target ficking of immune cells, which has a major impact on the is transcriptional repression or transcriptional activation in disease outcome (21) and opens a novel therapeutic avenue 2064 BRIEF REVIEWS: Bcl11b IN T CELLS AND IMMUNE DISEASES for and other Th17-mediated autoimmune How Bcl11b is integrated in such complex regulatory net- diseases. works remains to be further elucidated. As shown above, Pentao Liu’s group provided exciting ev- idence that the deletion of Bcl11b at early stages of thymic development or in DP thymocytes, with the use of an in- Disclosures ducible ER-Cre system, reprogrammed the T cells to NK-like The authors have no financial conflicts of interest. 2 2 cells. 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