Recent data illustrate a key role for the transcriptional regulator bric-a-brac, tramtrack, and broad complex and cap’n’collar homology (Bach)2 in orchestrating T cell differentiation and function. Although Bach2 has a well-described role in B cell differentiation, emerging data show that Bach2 is a prototypical member of a novel class of transcription factors that regulates transcriptional activity in T cells at super-enhancers, or regions of high transcriptional activity. Accumulating data demonstrate specific roles for Bach2 in favoring regulatory T cell generation, restraining effector T cell differentiation, and potentiating memory T cell development. Evidence suggests that Bach2 regulates various facets of T cell function by repressing other key transcriptional regulators such as B lymphocyte–induced maturation protein 1. In this review, we examine our present understanding of the role of Bach2 in T cell function and highlight the growing evidence that this transcriptional repressor functions as a key regulator involved in maintenance of T cell quiescence, T cell subset differentiation, and memory T cell generation.
Significant progress has been made in identifying and delineating the effects of key transcriptional regulators that govern the diverse fates of lymphocytic effector subsets. One such regulator is the transcriptional repressor bric-a-brac, tramtrack, and broad complex and cap’n’collar homology (Bach)2. In addition to its well-defined role in B cell and plasma cell differentiation (recently reviewed in Refs. 1–3), Bach2 is emerging as a functionally important regulator of other immune cell types, including macrophages and T cells (4). In this review, we discuss our present understanding of the role of Bach2 in regulating T cell development and homeostasis, as well as the emerging role of Bach2 in regulating the differentiation and function of effector and memory T cells. The potential for Bach2 to regulate various states of T cell activation, including quiescence and exhaustion, is also discussed.
Bach2 is a member of the Bach family of basic leucine zipper transcription factors (Fig. 1A). The Bach2 gene is located on human chromosome 6, 6q15 (chromosome 4, 4A4 in mouse), and encodes a 741-aa protein whose functional domains are highly conserved (>94%) in mice and humans (5, 6). Bach2 expression was originally described as being confined to the B cell lineage and to some neuronal cells that expressed a neuron-specific splice variant (7). However, Bach2 expression was later identified in the T cell lineage where it was reported to bind the IL-2 promoter and was required for maintenance of IL-2 production by human cord blood CD4+ T cells (8).
The basic leucine zipper transcription factors characteristically form heterodimers through their leucine zippers with the musculoaponeurotic fibrosarcoma (Maf) family of proteins yielding NF-E2 transcription factors (reviewed in Ref. 9). Bach2 forms heterodimers with small Maf proteins, including, MafF, MafG, and MafK, allowing binding to Maf recognition elements (MAREs) with the consensus sequence TGCTGA(G/C)TCA(T/C) (7) (Fig. 1B). Bach2 contains a nuclear localization signal in its Zip domain and a nuclear export signal at its C terminus. Several factors regulate Bach2 activity and localization (reviewed in Ref. 2), including PI3K signaling in B cells, which leads to phosphorylation of Ser512 and cytosolic accumulation. Oxidative stress inhibits the activity of the nuclear export signal and thus leads to nuclear accumulation.
Bach2 function has been most extensively investigated in B cells where it is known to repress expression of B lymphocyte–induced maturation protein 1 (Blimp-1), also known as PR domain zinc finger protein 1 (PRDM1), by binding to the MARE 5′ of the prdm1 (Blimp-1) gene (10). Conditional ablation of Bach2 in the B cell lineage has revealed that Bach2 downregulation is essential not only for Blimp-1 de-repression and differentiation of B cells into plasma cells, but also for class switch recombination leading to IgG1 secretion (11). However, the fates of B cells are not governed simply by Bach2 repression of Blimp-1. Rather, a complex transcription factor network controls memory B cell and plasma cell differentiation and key molecular events concomitant with differentiation, including class switch recombination, somatic hypermutation, and Ig secretion (1, 2).
Bach2 in disease
The bach2 gene locus is susceptible to modifications that impact health. Aberrations in the long arm of chromosome 6 are often associated with B cell malignancies. This includes a Bach2–Bcl2LI fusion product detected in a lymphoma line (12). This resulted in enhanced expression of the antiapoptotic protein Bcl2LI (also known as Bcl-xL). In another study, chromosomal rearrangements in an IgH-Myc–positive lymphoma resulted in fusion of exon 1 of IgHCδ on 14q32 to exon 2 of bach2 (13). The fusion transcript spanned the entire coding region (exons 2–9) of bach2 and was highly expressed. Thus, aberrant expression of Bach2 and/or its fusion partners is concordant with B cell malignancies, reinforcing the notion that Bach2 critically governs homeostasis in B lymphocytes.
The bach2 locus was shown to be a site of HIV-1 integration in resting CD4+ T cells in patients undergoing retroviral therapy (14). The bach2 locus may therefore constitute part of the mechanism by which patients with HIV infection maintain a reservoir of latently infected T cells. Bach2 expression changes also correlate with some diseases where aberrant T cell function is evident. For example, in patients with celiac disease, IFN-γ expression is strongly upregulated whereas Bach2 expression is strongly downregulated (15). Genome-wide association studies have revealed single nucleotide polymorphisms in the bach2 gene that are associated with a greater risk for developing rheumatoid arthritis (16), Crohn’s disease (17), multiple sclerosis (18), type I diabetes (19), and asthma (20). The bach2 polymorphisms observed were common to more than one disease, but often not equally associated. For example, the rs1847472 single-nucleotide polymorphism was strongly associated with celiac disease, but weakly associated with asthma. Therefore, bach2 polymorphisms are likely important contributors to susceptibility to different diseases, but they may have varying importance depending on the immune mechanisms driving pathology.
Bach2 regulation of T cell development and homeostasis
Originally described as a transcriptional regulator of B cells, it is now well appreciated that α/β T cells also express Bach2 (Fig. 2A). Bach2-encoding mRNA has been detected in thymic and peripheral T-lineage cells (21). Bach2 transcription gradually increases as T cells undergo differentiation and selection in the thymus, with more pronounced increases as T cells reach the single-positive developmental stage (21). Expression is additionally regulated upon thymic egress, at least in CD8 T cells, as Bach2 mRNA expression further increases in naive CD8 T cells as they seed the periphery (21). The factors regulating the expression of Bach2 in developing and mature T cells remain poorly defined, but TCR crosslinking via Ag encounter may lead to a reduction in Bach2 expression. Consistent with this, Ag-experienced effector and memory CD4 and CD8 T cells recovered from the periphery express lower levels of Bach2 transcripts, compared with their naive counterparts (21). As detailed below, cytokine signaling and other extrinsic factors are likely important for modulating Bach2 expression or activity in T cells. Furthermore, the expression of Bach2 is likely to be posttranscriptionally regulated in lymphocytes, as CD4 T cells express lower amounts of Bach2 protein compared with B cells, despite similar abundance of Bach2 transcripts (21). Recent data also established that the epigenetic regulator MENIN promotes Bach2 expression in CD4 T cells via maintenance of histone acetylation at the bach2 locus. This prevents T cell acquisition of a potentially damaging senescence-associated secretory phenotype, characterized in part by spontaneous proinflammatory cytokine production associated with loss of Bach2 expression (22), and further links Bach2 activity to the maintenance of T cell homeostasis. Delineating the mechanisms that govern Bach2 transcription and posttranscriptional regulation in T cells as they develop, mature, undergo homeostatic proliferation, and respond to infection will be critical to further elucidate the biological role and function of Bach2 in scenarios of both health and disease.
T cell expression of Bach2 is critical for the generation of regulatory T cells (Tregs). bach2−/− mice develop a progressive wasting disease, marked by an increased presence of antinuclear Abs associated with severely decreased numbers of Foxp3+ Tregs (23, 24). Expression of Bach2 may also be important for regulating the function of Tregs, as the limited number of Tregs recovered from bach2−/− mice exhibited a terminally differentiated phenotype and were unable to prevent disease in a T cell adoptive transfer model of colitis (23, 24). Bach2 also regulates the formation of peripherally induced Tregs (iTregs). The absence of Bach2 severely abrogated the generation of iTregs following activation in the presence of TGF-β, instead yielding activated T cells expressing a number of genes associated with other functionally distinct CD4 T cell subsets (23, 24). This includes a number of genes associated with T helper lineage commitment such as GATA-3, T-bet, and retinoic acid–related orphan receptor (ROR)γt. Thus, Bach2 may serve to allow for iTreg differentiation by preventing the expression of other key regulators of effector T cell differentiation. Intriguingly, Bach2 may also function differentially depending on the T cell developmental stage or maturation checkpoint, as Bach2 serves as a transcriptional inducer of IL-2 in human cord blood CD4 T cells (8). The factors that determine reported differential functions of Bach2 in CD4 T cells, and whether the functional impacts of Bach2 depend on the developmental stage of T cells, currently remain unknown and warrant further investigation.
Bach2 regulation of effector T cell differentiation and activity
Functionally distinct subsets of T cells regulate various aspects of immunologic protection against microbial infections, humoral immunity, and autoimmune pathogenesis. In addition to Tregs, emerging data support the concept that Bach2 plays a critical role in regulating the differentiation of functionally distinct effector T cells (Fig. 2B). A recent study identified Bach2 as the potential first member of a novel class of transcriptional repressors that limit transcriptional activity at genomic loci referred to as super-enhancers (SEs) (25). SEs are regions of enhanced transcriptional activity that are postulated to play essential roles in establishment of the functional identity of T cell lineages and subsets (26). For example, in peripheral T cells, SE regions are typically associated with genes that regulate lineage-specific cytokine responses (25). These include cytokine or cytokine receptor genes such as Ifng, Il17a,f, and Il4ra as well as lineage-determining transcription factor genes such as tbx21 (T-bet), gata3 (GATA-3), and rorc (RORγt) (25). Thus, as a repressor of transcription at SE regions, Bach2 can profoundly suppress the development and polarization of functional T cell responses. In support of this, whereas Bach2 is not required for T cell development in the thymus, it appears required to maintain quiescence of naive T cells in the periphery (21). Indeed, specific deletion of Bach2 in T cells leads to a spontaneous decrease in the number of phenotypically naive CD4 T cells (based on CD62Lhi and CD44lo expression patterns) and an increase of CD62L− cells that exhibit transcriptional profiles resembling those of effector memory T cells, in the absence of any pathogenic challenge (21). Importantly, although these data strongly support the idea that Bach2 functions to maintain T cell quiescence in the periphery, note that bach2−/−, CD62L− T cells did not develop into bona fide, functional Ag-specific effector memory T cells, as evidenced by their low expression of CD44. Additionally, despite their activated phenotype, bach2−/− T cells were less efficient than wild-type cells in protecting the host from Listeria monocytogenes infection or inducing colitis following transfer into rag1−/− recipient mice (21). Thus, bach2−/− T cells may not acquire full effector function, despite their activated phenotype. Rather, T cells lacking Bach2 appear to spontaneously develop an activated phenotype independently of antigenic encounter, suggesting that Bach2 actively restrains T cell activation at steady-state or during homeostatic, cytokine-driven turnover and maintenance. Thus, although Bach2 restrains T cell activation at steady-state, its absence is insufficient for T cells to develop the functional advantages and heightened protective capacity associated with effector or memory T cells.
In further support of the role of Bach2 as a regulator of T cell subset differentiation, bach2−/− CD4 T cells exhibited enhanced Th2 differentiation and enhanced Th2-associated cytokine expression (21). Notably, enhanced Th2 differentiation among bach2−/− CD4 T cells was apparent even in the presence of Th1-promoting cytokines that normally restrain Th2 differentiation, including IL-12 (21). One explanation for these observations is the capacity of Bach2 to suppress multiple genes that are linked to Th2 differentiation and function, including the transcription factor Blimp-1, which can potently suppress Th1 gene expression profiles (21, 27, 28). Thus, Bach2 can act as an important transcriptional regulator that facilitates CD4 T cell differentiation toward a Th1 phenotype and function via suppression of Th2 transcriptional programs. Similarly, Bach2 can restrain Th17 differentiation, potentially through the repression of the aryl hydrocarbon receptor (24), as aryl hydrocarbon receptor ligands strongly promote Th17 differentiation (29). Whereas the factors that regulate the expression of Bach2 to favor differentiation toward the various T helper subsets remain undefined, the key role for cytokines in dictating CD4 T cell subset differentiation strongly suggests that cytokines may also be involved in Bach2 regulation. It will be of tremendous interest to delineate how Bach2 expression is regulated during the course of T cell activation and to determine potential context-specific roles of Bach2 in the differentiation of functionally distinct CD4 T cells subsets, including other key CD4 subsets such as T follicular helper (Tfh) and Th9 cells.
The Tfh cell subset is essential for orchestrating the germinal center reaction, affinity maturation, Ig class switching, and the generation of memory B cells. Bcl-6 is identified as a key regulator of Tfh development and function, and similar to Bach2, Bcl-6 can function to suppress Blimp-1, an antagonist of both Th1 and Tfh development. Given the parallels between Bcl-6 and Bach2 in suppressing Blimp-1 expression, and the key role of Blimp-1 in repressing Tfh differentiation (30), it is interesting to speculate that Bach2 and Bcl-6 independently, simultaneously, or coordinately function to govern Tfh cell development. Indeed, at least in B cells, there is precedent for complex formation between Bach2 and Bcl-6 during repression of the promoter of prdm-1, the gene encoding Blimp-1 (31). Further work is required to dissect the specific contributions of Bach2 in regulating Tfh development, commitment, and activity.
Accumulating data support the idea that Bach2 also exerts profound effects on the function of fully differentiated, mature T cells. As noted above, studies show that bach2−/− mature T cells express significantly elevated levels of the Th2-associated cytokine IL-4 following activation compared with wild-type counterparts (21). These data are consistent with a role for Bach2 in limiting T cell activation and function. Alternatively, studies of bach2−/− Tregs reveal markedly impaired chemokine receptor expression, which may explain why Bach2-deficient Tregs can still suppress effector T cells in vitro (23), but bach2−/− Tregs fail to limit colitis in the naive CD4 T cell transfer models of colitis (23) or infiltrate tumors (32). Thus, although bach2−/− T cells generally exhibit enhanced effector function, loss or downregulation of Bach2 in T cells may limit their capacity to efficiently traffic to sites of inflammation or infection. More studies are required to fully elucidate the role of Bach2 in regulating T cell homing, trafficking, and functional activities that extend beyond elaboration of cytokines during health and disease.
Bach2 regulation of memory T cell development and function
Recent studies are establishing an important role for Bach2 in shaping the development and biology of memory T cells (Fig. 3). Using bioinformatics approaches, Hu and Chen (33) mined 386 publicly deposited gene expression databases detailing the transcriptional profiles of CD8 T cells in different states of activation. This analysis led to the identification of a number of transcription factors not previously associated with memory CD8 T cell memory generation, including Bach2 (33). The biological and functional relevance of Bach2 in memory T cell progression was demonstrated by retroviral-mediated transduction of Bach2 in CD8 T cells, which resulted in increases in the number of CD8 T cells at the peak of the response as well as increased survival during the contraction phase to establish memory populations (33). Similarly, Restifo and colleagues (34) recently demonstrated that bach2−/− CD8 T cells fail to accumulate or form numerically stable memory pools after acute viral infection. Additionally, they demonstrated that Bach2 restrains CD8 T cell effector differentiation by interfering with the function of a subset of AP-1 family transcription factors (34). Taken together, these genetic studies confirm that Bach2 is functionally linked to the establishment of memory potential in T cells.
Although the transcriptional networks governing the induction and effects of Bach2 on memory T cell development have not been fully elucidated, repression of Blimp-1 expression by Bach2 is likely a critical component of the mechanism by which Bach2 promotes memory T cell formation. Blimp-1 expression is required for the differentiation of short-lived effector cells, and Blimp-1 deficiency favors the development of memory T cells (35, 36). Indeed, Blimp-1 deficiency in T cells promotes the acquisition of memory characteristics among effector T cell populations (36). Thus, Bach2-mediated enhancement of memory CD8 T cell generation is likely linked to its capacity to downregulate the expression of genes associated with effector differentiation, thereby tipping the balance in favor of generating memory cells earlier in the response. This is supported by recent data demonstrating that Bach 2 can compete with AP-1 family transcription factors to prevent the induction of the effector transcriptional program in CD8 T cells (34). Notably, XBP-1, which is classically regarded as a critical regulator of Ab secretion by B cells (37, 38), has also been linked to promoting the terminal differentiation of T cells via activation of endoplasmic reticulum stress (39). In B cells, the transcriptional control circuit of XBP-1 splicing and activation is in turn regulated by Blimp-1, whose activity is further repressed by Bach2. It is therefore likely that many of the same transcriptional networks operating in B cells also impact the fate, function, and survival of effector and memory T cells. Given the intimate functional links between the activity of Bach2 and the expression and function of Blimp-1, it is plausible that Bach2 functions upstream of pathways that promote the formation and maintenance of long-lived T cell immunity following infection or vaccination.
The development of memory characteristics in CD8 T cells has been postulated to represent a default pathway that gets diverted toward effector CD8 T cell development and acquisition of effector function by inflammatory cytokines (40) (Fig. 3). Recent data show that specific inflammatory cytokines and innate signals, including IL-12/IL-18 or IL-15, as well as TLR signaling, promote microRNA (miRNA) miR-181a and miR-148a expression in human and murine leukocytes (41, 42), and miR-148a has been shown to directly limit Bach2 expression (43). Altered regulation of Bach2 may function as a mechanistic link by which specific inflammatory cytokines and miRNA regulate T cell differentiation, survival, and memory formation. Consistent with this hypothesis, overexpression of miR-181a in T cells enhances expression of costimulatory molecules and potentiates calcium flux following TCR crosslinking (44). Both the quality and duration of TCR signaling regulate memory T cell progression (45–48). Thus, miRNA-mediated gene regulation represents a critical additional level of posttranscriptional genetic control with profound effects on gene expression in immune cells. Moreover, the cytokine-induced activity of specific miRNAs targeting Bach2 would also be consistent with data demonstrating that Bach2 expression is posttranscriptionally regulated in distinct lymphocyte subsets (21). It is possible that Bach2 actively suppresses T cell effector functions (favoring the default memory pathway) and that T cell exposure to inflammatory cytokines is required to reduce its expression allowing for the establishment of T cell transcriptional effector programs and enhanced effector function. Collectively, these studies further support the hypothesis that Bach2 functions at the nexus of a critical transcriptional network that governs effector and memory T cell differentiation, activity, and survival.
Bach2 as a potential regulator of T cell exhaustion?
Following acute infection or vaccination, Ag-specific T cell populations undergo proliferative expansion, acquire and exert effector functions, and subsequently undergo numerical contraction leaving pools of memory T cells that afford the host enhanced protection against reinfection (49). Similarly, T cells responding to chronic infections in which the pathogen is not efficiently eliminated also undergo similar phases of expansion and contraction. However, unlike scenarios of acute infection or vaccination, T cells responding to persistent infection exhibit specific alterations in gene expression profiles that are associated with functional impairments, including loss of cytokine expression, reduced T helper and cytolytic potential, and increased rates of apoptosis (50, 51). These biological processes are collectively known as T cell exhaustion, which is phenotypically, functionally, and molecularly distinct from T cell anergy or senescence (reviewed in Ref. 52). The molecular mechanisms responsible for T exhaustion are multifactorial and include perturbations in the transcriptional programming of T cells.
Regarding altered expression and function of transcription factor networks, Eomes (53), T-bet (54), and Blimp-1 (55) promote T cell exhaustion, whereas Id3 (56) limits T cell terminal differentiation and exhaustion. Given the capacity for Bach2 to regulate these transcription factor networks, it is tempting to speculate that Bach2 may critically govern T cell exhaustion. In support of this hypothesis, recent profiling studies of exhausted CD4 and CD8 T cells responding to chronic viral infection showed that Bach2 expression is reduced 2- to 4-fold compared with conventional (i.e., functional) memory CD4 and CD8 T cell subsets (57). Repeated TCR stimulation may also be important for potential reductions in Bach2 expression in T cells responding to chronic infection or persistent Ag. As noted above, effector and memory T cells express relatively reduced Bach2 compared with naive T cells (33), and as T cells are driven through sequentially progressive recall responses to generate secondary, tertiary, and quaternary memory cells, Bach2 expression is proportionately reduced (58). Interestingly, these multiply stimulated memory T cells maintain a more effector-like profile, suggesting that this may be controlled by progressive loss of Bach2 following repeated TCR triggering. As a composite, these data are consistent with an important mechanistic role for Bach2 in limiting terminal differentiation of T cells, preventing T cell exhaustion, and promoting long-lived numerically and functionally stable populations of memory T cells.
T cells are essential for providing protection against intracellular pathogens and orchestrating humoral immunity. Conversely, T cells can also be pathologic, driving autoimmune disease, allergy, or atopy. Thus, understanding the molecular mechanisms that govern the development, differentiation, and function of Ag-specific T cells remains a critically important goal. Bach2 is emerging as a fundamentally important molecular switch that can limit terminal differentiation of T cells and constrain their functional activity, potentially promoting the generation of long-lived, highly functional memory T cell populations. Despite this, little is known about the pathways that regulate Bach2 expression in T cells. It will be of significant interest to determine whether Bach2 is mechanistically linked to limiting or preventing T cell exhaustion or senescence. Discovering new circuits of molecular control of T cell differentiation, function, and memory formation will facilitate the development of new approaches to maintain immune homeostasis, limit pathological T cells responses, and enhance T cell vaccine-induced protective immunity.
We thank Dr. S. A. Condotta (McGill University) for generating the figures and Dr. L. Thompson (Oklahoma Medical Research Foundation, Oklahoma City, OK), Dr. C. Webb (University of Oklahoma Health Sciences Center), and Dr. S. A. Condotta for critical reading of the manuscript.
M.J.R. is supported by funds from the Canada Foundation for Innovation (John R. Evans Leaders Fund) and by National Sciences and Engineering Research Council of Canada Grant RGPIN-2016-04713. N.S.B. is supported by National Institutes of Health/National Institute of Allergy and Infectious Diseases Grant AI125446 and National Institutes of Health/National Institute of General Medical Sciences Grant GM103447. M.L.L. is supported by National Institutes of Health/National Institute of Allergy and Infectious Diseases Grant AI078993 and by funds from the Presbyterian Health Foundation of Oklahoma City.
Abbreviations used in this article:
bric-a-brac, tramtrack, and broad complex and cap’n’collar homology
B lymphocyte–induced maturation protein 1
peripherally induced regulatory T cell
Maf recognition element
PR domain zinc finger protein 1
retinoic acid–related orphan receptor
regulatory T cell
T follicular helper.
The authors have no financial conflicts of interest.