Decades of research have demonstrated the essential role of E proteins in mediating both innate and adaptive immune cell development and the wide implications of E protein activity in disease progression and immune response. In mammals, E proteins include E2A, E2-2 and HEB (1). E proteins are members of the helix-loop-helix (HLH) family of transcription factors. E proteins either homo- or heterodimerize with other HLH proteins to bind to E-box sites (CANNTG) through a basic region to modulate the expression of nearby and distal genes. This activity is opposed by Id proteins, which lack a basic DNA binding region and heterodimerize with E proteins to prevent them from binding to DNA. Together, E and Id proteins form an E-Id axis to instruct immune development.

This review discusses the role of the E-Id axis in adaptive immune development. These proteins are expressed in all mammalian cell types. They are regulated both transcriptionally and post-transcriptionally to orchestrate the development of an armamentarium of immune cell types and to establish a diverse immune repertoire. We focus on how appropriately timed differentiation to T and B cell fates is achieved while discussing how the development of alternative cell fates is suppressed.

Adaptive immune development begins in the fetal liver and in the bone marrow in adults, where E and ID proteins influence developmental decisions in hemopoietic stem cells (HSCs), which give rise to all blood cells ( Figure 1 ). Differentiation to HSCs is achieved by the E protein SCL/TAL1, and maintained by E2A proteins and their repressive heterodimerizing HLH partners Lyl1 and Id1 (2–5). These factors also set the stage for the ratio of progenitors giving rise to B and T cells. E proteins oppose proliferation of HSCs, priming their expression to promote lymphoid-associated gene expression (6, 7). This activity promotes their differentiation into multipotent progenitors (MPPs) and further into lymphoid-primed MPPs (LMPPs), while preventing granulocyte-monocyte progenitor (GMP) development and partially restricting megakaryocyte-erythrocyte progenitor (MEP) development (6). As a result, E2A-deficient mice are associated with reduced HSCs and MPPs (3). Other E proteins, E2-2 and HEB, were found to be expendable at this early stage of development (8–10). Thus, E protein activity orchestrates a supportive transcriptional landscape for lymphocyte development in HSCs.

Id protein inhibition of E protein activity might play a role in generating a diverse immune repertoire from HSCs. E2A promotes HSC differentiation and represses proliferation by controlling the expression of p21 and Notch1 (11–14). E proteins further drive differentiation to LMPPs to common lymphoid progenitors (CLPs), which give rise to several cell fates including B cells, T cells, dendritic cells, innate lymphoid cells, and natural killer (NK) cells (10, 15). In the absence of E2A expression, fewer LMPPs progress to the CLP stage (6). The cells that do progress preferentially feed alternative lineages seeded by GMPs, MEPs, and Pre-MegE-progenitors (7). In the absence of E2A and HEB, CLPs are compromised in their ability to express an early lymphoid program (10). The capacity of CLPs to differentiate into NK, B or T lineages may be further divided by their expression of different surface markers (16–19). A recent study suggested that heterogeneous levels of E and Id proteins in CLPs may contribute to these unique differentiative capabilities (19). Thus, fine tuning of E protein activity in CLPs instructs immune cell fate.

E proteins orchestrate B cell development by defining signaling pathways in CLPs ( Figure 2 ). E2A activity is required to activate Ebf1 and IL7 receptor protein (IL7Rα) in CLPs, which together with E2A activate Pax5 (20–23). E2A proteins also act in concert with Ebf1 to induce Foxo1 expression (24). Subsequently, E2A and HEB coordinate with Foxo1, Pax5 and Ebf1 to support the progression of CLPs through the B cell lineage (23, 25). Aberrant Id3 expression at this earlier stage alternatively arrests B cell development and prevents IL7Rα induction, later inducing caspase-mediated apoptosis (26). Cytokine signaling from the TGF-β family represents one mechanism by which Id3 expression is regulated (27). Thus, E proteins promote B cell development from CLPs by priming a B-cell transcriptional network.

E proteins then stimulate the subset of CLPs primed with B cell lineage genes to progress into pre-pro-B cells. Studies have implicated that the E2A isoform E47 is required to get past this stage, while E12 is dispensable (28–31). E2-2 and HEB also both contribute to early B cell development (32). Notably, E2-2 mRNA expression is particularly high in pro-B cells and orchestrates the developmental maturation of these early B cell progenitors into pre-B cells (33).

In the B cells, E proteins are regulated by lineage specific post-transcriptional mechanisms. E47 homodimers, for example, are only detected at high levels in B cells (34). This cell specific homo-dimer activity may involve phosphorylation of specific residues of the E2A proteins (34, 35). Histone acetyltransferases including p300, CBP, and PCAF interact with E2A to mark the epigenetic chromatin landscape (36, 37). Further, miRNAs regulate E protein activity. A recent study identified miR-191 as a rheostatic regulator of B cell development to modulate E2A mRNA abundance from pro-B to immature B cells (38).

E proteins regulate gene expression by coordinating changes in nuclear architecture. E2A occupancy at the Ebf1 locus is associated with relocation away from the nuclear lamina in pro-B cells (39). E2A binding at or near enhancers or promoters was associated with deposition of active chromatin markers such as H3K4me1 with further activating epigenetic alterations between pre-pro-B to pro-B cells (40). Activated genes with E2A occupancy frequently contained coordinated DNA binding with Ebf1, Foxo1 and CTCF. Recent studies indicated that E2A occupancy is closely associated with recruitment of members of the cohesin complex (41). In parallel studies it was revealed that H3K27Ac marked enhancers are closely associated with recruitment of cohesin (42–44).

Collectively, these studies suggest that E2A may act, at least in part, by initiating loop extrusion across the enhancer landscape. Other mechanisms may also act with E2A to promote B cell development. Notably, E2A recruits Tet2 and Tet3 to promote chromatin accessibility adjacent to E-box binding sites in pro-B cells (45). Future studies are warranted to determine how E2A mediated changes in DNA methylation and recruitment of cohesin are linked to induce lineage specific gene programs.

E proteins may also be essential in preventing the premature progression of B cells from pre-pro-B into pro-B cells. E2A enforces this checkpoint by binding an E-box element in a p21 regulatory region, which encodes a CDK inhibitor and induces cell cycle arrest (11). This checkpoint can be circumvented by repression of E protein activity by Id1-3 (11, 26). The downregulation of E protein activity by rapid induction of Id proteins occurs upon successful heavy chain V-DJ rearrangement in pro-B cells, and it will be important to establish whether and how alterations in Id3 protein levels modulate E2A activity to orchestrate antigen receptor assembly (28).

Somatic recombination events in the B cell lineage generate a diverse antibody repertoire. B cells rearrange the variable regions of their immunoglobulin heavy chain (Igh) and immunoglobulin light chain (Igk or IgL) loci to produce mature B cells with unique antigen binding specificities. E2A regulates these recombination events by controlling appropriate expression of the Rag genes, as well as chromatin accessibility and 3D spatial organization of the Igh and Igk loci. Following these recombination events, B cells can undergo class switch recombination (CSR) and somatic hypermutation (SHM) to generate antigen receptors with higher affinities for their cognate antigens. E proteins regulate CSR and SHM by promoting chromatin accessibility at the targeted immunoglobulin (Ig) genes and by controlling the expression of key enzymes involved in these processes.

After successful VDJ rearrangement and receptor editing of the Ig light chain genes, E and Id proteins further instruct the development of pre-B cells. This transition is mediated by upregulation of Id3 and a reduction in E protein abundance triggered by BCR signaling (49). E47 levels therefore decline in transitional B cells followed by a near complete loss of E47 expression in mature B cells. Genetic studies showed that high levels of E2A promote follicular B cell development while high Id3 abundance favors the marginal zone B cell fate (33, 49). E2-2 serves an overlapping role controlling this developmental decision as revealed by the transfer of E2A- and E2-2-deficient fetal liver cells into irradiated Rag-deficient mice (33). E protein activity is also essential for the development of germinal center and plasma cells (83). Likewise, in the absence of Id3 expression germinal center B cell development is severely affected (84). Specifically, when researchers abrogated Id3 expression in germinal center B cells, the expression of genes encoding for components of antigen receptors, cytokine receptors, and chemokine receptors was severely perturbed (83). E2A and E2-2 activity is also essential for the developmental progression of plasma cells (29, 84, 85). E2A and E2-2 promote plasma cell identity by directly activating Blimp1 and Xbp1 expression (84, 85). Together these studies show that HLH proteins play instrumental roles in orchestrating the response of B cells to exposure of infectious agents.

A fraction of LMPPs develops into early T progenitors (ETPs) that then home to the thymus ( Figure 4 ). E2A and HEB promote homing by modulating chemokine receptor expression, including CXCR4, to direct thymocytes to the cortex (100). Here, ETPs encounter Delta-class Notch ligands. An ensemble of genes involved in Notch signaling are directly activated by E47 (21). Together with E2A, Notch signaling prevents the activation of B-lineage and myeloid factors and promotes T lineage development. These functions are opposed by Id1 and Id2 expression in these early progenitors and in double negative (DN) T cells, promoting an innate lymphoid fate instead. T cell progression was blocked in CLPs with disrupted E2A and HEB activity, instead favoring differentiation to alternative lineages (101). Once ETPs migrate to the thymus, E2A and HEB, in coordination with Notch signaling instruct further development (21, 32, 102–104). In developing thymocytes, E proteins modulate the expression of gene programs including those involved in cell cycle progression, pre-TCR signaling and cytokine gene expression (105). Prominent amongst the genes activated by E47 expression are CDK6, Socs1/2, Ets, Foxo1, and GATA3. The E2A proteins may act coordinately with Bcl11b, another key factor known to establish T cell identity, to activate a common set of target genes (106, 107). Researchers have found that E2A binds regulatory elements in a distal long non-coding RNA, ThymoD (108). ThymoD transcription is initiated from within the Bcl11b intergenic region where it acts to promote T cell commitment by repositioning the Bcl11b enhancer from a heterochromatic environment at the lamina to the euchromatic compartment located in the nuclear interior (107, 108). The overlapping gene expression profiles between E2A, Bcl11b, and ThymoD knockout mice combined with the evidence of E2A binding to elements within ThymoD implicates the possibility that E2A could indirectly initiate Bcl11b expression by modulating non-coding transcription. A prominent Bcl11b target in developing thymocytes is Id2. Interestingly, the majority of genes regulated by Bcl11b are also modulated by Id2 (104, 109). HEB also performs multiple, unique functions in thymocyte development. Elegant studies revealed that an alternatively spliced form of HEB, named HEBAlt, increases the development of T cell progenitors (110, 111). Subsequent studies showed that in the absence of HEB T cell progenitors adopt alternative cell fates (112). HEB also directly activates the expression of pre-Tα, a component of the pre-TCR complex (113). Thus, a detailed picture is now emerging in which E2A and HEB act collaboratively to orchestrate the development of early T cell progenitors.

T lymphocytes rearrange their TCRα, TCRβ, TCRγ and TCRδ loci to generate diverse T cell receptor repertoires. T cell receptor rearrangements initiate at the CD4^- CD8^- double negative (DN) stage of thymocyte development. TCRβ, TCRγ and TCRδ rearrangements all occur simultaneously in DN2 cells. Successful rearrangement of TCRγ and TCRδ loci results in expression of a γδ TCR and potential development into the γδ T cell lineage. Successful rearrangement of a TCRβ chain results in expression of a pre-TCR. Pre-TCR signaling allows progression to the DN4 cell stage where TCRα rearrangements occur, so that cells may become αβ T cells. The E-ID axis plays critical roles in T cell development by regulating rearrangement events at all four TCR loci, orchestrating the αβ versus γδ cell fate decision, and by enforcing key developmental checkpoints.

While Id3 is generally involved in orchestrating γδ cell fate, the Id proteins restrict development of a specific subset of γδ T cells, γδ NKT cells. γδ NKT cells are innate-like γδ T cells that express a semi-invariant receptor (Vγ1.1Vδ6.3), and are associated with many innate like characteristics. Loss of Id3 expression in γδ T cells leads to higher E protein activity, upregulation of Egr2, PLZF, and c-Myc and proliferative expansion of γδ NKT cells (154). Id3 deficient mice also show an expanded population of γδ NKT cells (155–157). Id2 either cooperates with or can compensate for Id3, and γδ NKT cells are expanded even more so in Id3 deficient mice that also have compromised Id2 function (157). Deletion of Id2 promotes a smaller expansion of γδ T cells, although interestingly, this expansion of γδ is not limited to cells expressing Vγ1.1Vδ6.3 (157). Id2 and Id3 restrict development into the NKT γδ T cell fate by inhibiting E protein activity, and deletion of E proteins in Id deficient mice reverts the expansion of NKT γδ T cells (156, 157).

The mechanism by which inhibition of Id protein activity expands the γδ NKT population is unclear. It is possible that γδ NKT expansion could be a result of increased rearrangement, however there are conflicting findings regarding whether the expansion of Vγ1.1 γδ NKT cells in Id3 deficient mice occurs at the expense of other γδ T cells. It has been reported that Id3^-/- mice have reduced numbers of Vγ2 and Vγ3 dendritic epidermal T cell (DETC) subsets (116). Others note that the expanded use of the Vγ1.1 gene is not at the expense of other Vγ genes, and that the number of cells expressing Vγ2 and Vγ5 genes is the same, though the proportion of γδ T cells expressing them is reduced (158). One possible explanation for the expansion of γδ NKT cells is that these cells are normally deleted as a result of excessive γδTCR signaling, but Id3 deficiency allows them to escape deletion and proliferate (159).

A small fraction of DP thymocytes differentiate into invariant NKT (iNKT) cells, driven by heightened E protein activity and modulation of Id2/3 protein expression (160). Upon positive selection, iNKT cells further mature into multiple subsets, including NKT1, NKT2 and NKT17 cells. These developmental transitions are again instructed by E-Id protein activity to indirectly impact CD8+ T cell fate (161–165). iNKT cells express an invariant TCRα chain composed of the distally located V_α14-J_α18 gene segments, which recombine in secondary TCRα rearrangements. Several rounds of Vα to Jα recombination occur during TCRα rearrangement. Primary rearrangements of the TCRα locus make use of the most proximal 3’ Vα genes and most distal 5’ Jα genes. Secondary rearrangements make use of more 5’ Vα and 3’ Jα segments. Recombination is terminated when cells either pass positive selection or undergo cell death. Prolonged survival at the DP stage allows cells to undergo more sequential arrangements before undergoing cell death. HEB cooperates with TCF-1 to promote the survival of DP thymocytes by positively regulating the anti-apoptotic gene Bcl-XL (118, 166–169). DP thymocytes which lack HEB have an impaired ability to survive, rearrange their distal Jα genes less, and completely lack iNKT cells. This loss of iNKT cells is attributed to the shortened lifespan of DP cells and subsequent deficiencies in secondary TCRα rearrangements, as ectopic expression of Bcl-XL restores secondary TCRα rearrangements and iNKT development (118). Taken together, these data indicated that HEB instructs the generation of a diverse αβ T cell repertoire, enabling usage of all distally located genes and the development of iNKT cells ( Figure 5 ).

E protein activity mediates the development of DP thymocytes into unique developmental fates within the CD4+ or CD8+ T cell compartments. The role of E2A and HEB in supporting the development of an appropriate ratio of CD4+ to CD8+ T cells is now well established (170–172). These E proteins bind to CD4 E-box site to support CD4+ development while antagonizing Id2/3 activity is required for CD8+ development (171–173). E proteins mediate this development by modulating CCR7 and IL7Rα expression (171). Conversely, this activity is suppressed by Id proteins to guide CD8+ development (171). After the successful rearrangement of the TCRβ and TCRα loci, TCR-signaling induces Id3 expression, which is then maintained to enforce a naïve state in peripheral T cells (174). Id2 is then upregulated at a later stage through an unknown pathway. Id2 was found to downregulate Id3 while Id3 had no effect on Id2 expression, indicating that some of the Id2-mediated effects on gene expression may be indirect (175). In summary, sustained sequestration of E proteins by Id proteins may maintain thymic single positive T cells in a naïve state.

CD4+ T cells are instructed towards unique developmental fates by the delicate balancing and timing of E protein activity. Unopposed E protein activity readily leads to the development of innate variant follicular helper T cell (T_FH) cells (174). In peripheral CD4+ T cells, Id2 and Id3 act to support the Th1 development while restraining T_FH lineage differentiation (176). In parallel studies it was shown that Id2 suppresses T_FH development and expansion by activating the PI3K–AKT–mTORC1–Hif1a and c-myc/p19Arf pathways (173, 177). An alternative pathway that underpins T_FH cell development may involve the induction of Bcl6 expression, which in turn inhibits Id2 expression (176). By permitting T_FH development, E proteins also influence the formation of germinal centers, with higher amounts of GC and PC B cells found in both thymi and peripheral lymphoid organs derived from mice that harbor Id2/3 deletions (173, 174). These findings highlight the importance of E protein activity in T cells in coordinating a B cell response and for germinal center adaptive immune cell development. Id proteins also orchestrate developmental progression of Treg cells (178, 179). Upon depletion of Id2 and Id3 expression in Treg cells mice readily develop Th2-cell mediated inflammatory disease (178–180). Collectively these studies revealed that E and Id proteins modulate the development of an ensemble of distinct peripheral CD4+ T cells to combat infection and suppress the development of autoimmune disease.

Id2 and Id3 also regulate E protein activity to instruct CD8+ T cell development. Naïve CD8+ T cells stimulated by the appropriate antigen readily elevate E-protein DNA binding (181). A series of elegant studies revealed that the E-Id protein axis also controls the developmental progression of CD8+ effector and memory T cells (175, 182–186). High levels of Id2 expression are required to instruct CD8+ effector T differentiation while suppressing the development of CD8+ memory cells (182, 183, 185). Conversely, upregulated Id3 expression promoted the development of Cd8+ memory cells (175). Id proteins further perform a key role in orchestrating the development of long-lived resident memory (Trm) cells (187). In summary, E and Id proteins play critical roles in orchestrating the development of an ensemble of immune cell types that act collectively to combat infection.

Very early studies revealed that E47 protein abundance is noisy in naïve B cells. While a small proportion of naïve B cells express detectable levels of E47, E47 abundance is uniformly high in activated B cells (90). Consistent with these observations, more recent studies showed that E47 mRNA abundance varied across the naïve B cell population while heterogeneity in E47 mRNA levels in activated B cells was low (188). These findings raised the question as to how such differences in mRNA abundance and heterogeneity are established. Quantitative studies have addressed this question (188). E2A and E2-2 bursting frequencies and mRNA life-times differ between naïve and activated B cells. In naïve B cells, E2A and E2-2 bursting frequencies are low and mRNA life-times are short. Conversely in activated B cells E2A and E2-2 bursting frequencies are high and mRNA life-times are long (188). These findings bring into question how alterations in E2A and E2-2 mRNA life-times are established. One possible mechanism involves miRNA instructed fine-tuning of E2A and E2-2 mRNA abundance, and it will be important to identify potential miRNAs that target HLH genes. Finally, we would like to consider a role for heterogeneity in E2A and E2-2 mRNA abundance in instructing lymphocyte activation. We suggest that upon interacting of the BCR with invading pathogens, E2A and E2-2 heterogeneity in mRNA abundance permits a swift and clonal response. In such a scenario, the few B naïve cells that are actively bursting across the B cell population are primed to readily undergo CSR or rapidly develop into differentiating plasma cells. Conversely, increased E2A and E2-2 bursting frequencies and lower mRNA decay rates in activated B cells may decrease heterogeneity in E2A and E2-2 abundance to orchestrate B cell maturation. We propose that similar mechanisms instruct the immune response in T cells. Upon viral or tumor encounters the decision to differentiate into effector or memory T cell fate is similarly dictated by the combined alterations in E2A, E2-2 and HEB bursting frequencies and mRNA life-times.

Over three decades of research have highlighted critical functions of E- and Id-proteins in instructing adaptive immune development. E-proteins activate B- and T-lineage specific gene programs to specify B and T cell fate. They promote the assembly of antigen receptor loci to generate a diverse antibody and TCR repertoire. In maturing thymocytes, the E- and Id-proteins promote thymocyte selection. In peripheral B and T cells, the rise and fall in E- and Id-proteins orchestrate the development of an array of regulatory, effector and memory cell types. In mechanistic terms, E-proteins sequester histone acetyltransferases across the enhancer landscape to promote the deposition of H3K27Ac. The deposition of H3K27Ac, in turn, initiates loop extrusion to assemble a wide ensemble of loops across antigen receptor loci and down-stream target genes. We suggest that these proteins also assemble loop domains into nuclear condensates to regulate antigen receptor loci rearrangement and lineage specific programs of gene expression. Finally, we propose that alterations in HLH bursting frequencies and mRNA life-times increase and/or narrow heterogeneity in mRNA abundance to establish B or T cell identity, thereby instructing the developmental progression of peripheral effector and memory lymphocytes in response to invading pathogens.

MA and ZW wrote the manuscript and designed figures with inputs from CM. All authors contributed to the article and approved the submitted version.

ZW was supported by a David V. Goeddel Endowed Graduate Fellowship and by a training grant from the NIH (2T32DK007541-31A1). CM was supported by the NIH AI00880, AI09599, AI102853 and AI102853.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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