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E-protein transcription factors limit group 2 innate lymphoid cell (ILC2) development while promoting T cell differentiation from common lymphoid progenitors. Inhibitors of DNA binding (ID) proteins block E-protein DNA binding in common lymphoid progenitors to allow ILC2 development. However, whether E-proteins influence ILC2 function upon maturity and activation remains unclear. Mice that overexpress ID1 under control of the thymus-restricted proximal Lck promoter (ID1tg/WT) have a large pool of primarily thymus-derived ILC2s in the periphery that develop in the absence of E-protein activity. We used these mice to investigate how the absence of E-protein activity affects ILC2 function and the genomic landscape in response to house dust mite (HDM) allergens. ID1tg/WT mice had increased KLRG1− ILC2s in the lung compared with wild-type (WT; ID1WT/WT) mice in response to HDM, but ID1tg/WT ILC2s had an impaired capacity to produce type 2 cytokines. Analysis of WT ILC2 accessible chromatin suggested that AP-1 and C/EBP transcription factors but not E-proteins were associated with ILC2 inflammatory gene programs. Instead, E-protein binding sites were enriched at functional genes in ILC2s during development that were later dynamically regulated in allergic lung inflammation, including genes that control ILC2 response to cytokines and interactions with T cells. Finally, ILC2s from ID1tg/WT compared with WT mice had fewer regions of open chromatin near functional genes that were enriched for AP-1 factor binding sites following HDM treatment. These data show that E-proteins shape the chromatin landscape during ILC2 development to dictate the functional capacity of mature ILC2s during allergic inflammation in the lung.

The response of Innate Lymphoid Cells (ILCs) to conditioning regimens and subsequent recovery in the setting of allogeneic hematopoietic stem cell transplant (aHSCT) in pediatric patients is not known. The impact of this recovery pattern on graft versus host disease (GVHD) has not been prospectively studied. We aim to study the recovery of ILCs after aHSCT in children and discover its role in GVHD. Pediatric patients undergoing aHSCT were enrolled in a prospective translational study since May 2019. Patients blood were analysed by flow cytometry at following time points: prior to the conditioning regimen, weekly starting on day 0 until +30 (+/− 3 days), then +60, +90 and +180 days. Ten patients have been enrolled to date - demographics: median age 9.5 years; 4 malignant, 6 non-malignant; haploidentical donor (n=1), matched sibling (n=3), unrelated (n= 6). Graft source was bone marrow for all patients. Most common conditioning regimen was fludarabine, busulfan and rabbit antithymocyte globulin (40%). GVHD prophylaxis for most patients was tacrolimus with mini-methotrexate (90%). We saw substantial drop in number of ILCs in response to conditioning regimen in all patients, with nadir on +14 that plateaued through +30, followed by a spike on +60 and return to normal levels by +90. Similar trend was seen in type 1 ILCs (Lin− CD127+ CRTH2− CD117−) but showed a slower drop, reaching nadir on +21 with slow recovery by +60. Type 2 ILCs (Lin− CD127+ CRTH2+) and Type 3 ILCs (Lin− CD127+ CRTH2-ve CD117+) however showed a small increase on +7 compared to day 0 with both cell types recovering by +60. We showed that it is feasible to prospectively study the recovery of ILCs after aHSCT. Continued enrollment will allow us to evaluate the correlation between ILCs and GVHD.

E protein transcription factors and their natural inhibitors, Id proteins, play critical and complex roles during lymphoid development. In this article, we report that partial maintenance of E protein activity during positive selection results in a change in the cell fate determination of developing iNKT cells, with a block in the development of iNKT1 cells and a parallel increase in the iNKT2 and iNKT17 subsets. Because the expression levels of the transcription factors that drive these alternative functional fates (GATA-3, RORγT, T-bet, and Runx-3) are not altered, our results suggest that E protein activity controls a novel checkpoint that regulates the number of iNKT precursors that choose each fate.

Notch signaling pathway plays important roles in promoting the generation of marginal zone (MZ) B cells at the expense of follicular (FO) B cells during periphery B cell maturation, but the underlying molecular mechanisms are not well understood. We hypothesize that Notch favors the generation of MZ B cells by downregulating E protein activity. In this study, we demonstrated that expression of Id2 and ankyrin-repeat SOCS box-containing protein 2 was elevated in MZ B cells and by Notch signaling. Id2 inhibits the DNA binding activity of E proteins, whereas ankyrin-repeat SOCS box-containing protein 2 facilitates E protein ubiquitination. Next, we examined the phenotypes of splenic B cells in mice expressing constitutively active Notch1 and/or two gain-of-function mutants of E proteins that counteract Id2-mediated inhibition or Notch-induced degradation. We found that upregulation of E proteins promoted the formation of FO B cells, whereas it suppressed the maturation of MZ B cells. In contrast, excessive amounts of Notch1 stimulated the differentiation of MZ B cells and inhibited the production of FO B cells. More interestingly, the effects of Notch1 were reversed by gain of E protein function. Furthermore, high levels of Bcl-6 expression in FO B cells was shown to be diminished by Notch signaling and restored by E proteins. In addition, E proteins facilitated and Notch hindered the differentiation of transitional B cells. Taken together, it appears that Notch regulates peripheral B cell differentiation, at least in part, through opposing E protein function.

Multiple subsets of the bone marrow contain T cell precursors, but it remains unclear which is most likely to replenish the adult thymus. Therefore, RAG-1+ early lymphoid progenitors (RAG-1+ ELP), and CD62L/L-selectin+ progenitors (LSP), as well as common lymphoid progenitors from C57BL6-Thy1.1-RAG-1/GFP mouse bone marrow were directly compared in transplantation assays. The two c-Kithigh populations vigorously regenerated the thymus and were superior to common lymphoid progenitors in magnitude and frequency of thymic reconstitution. Regeneration was much faster than the 22 days described for transplanted stem cells, and RAG-1+ ELP produced small numbers of lymphocytes within 13 days. As previously reported, LSP were biased to a T cell fate, but this was not the case for RAG-1+ ELP. Although RAG-1+ ELP and LSP had reduced myeloid potential, they were both effective progenitors for T lymphocytes and NK cells. The LSP subset overlapped with and included most RAG-1+ ELP and many RAG-1−TdT+ ELP. LSP and RAG-1+ ELP were both present in the peripheral circulation, but RAG-1+ ELP had no exact counterpart among immature thymocytes. The most primitive of thymocytes were similar to Lin−c-KithighL-selectin+TdT+RAG-1− progenitors present in the marrow, suggesting that this population is normally important for sustaining the adult thymus.

Dendritic cells (DC) comprise plasmacytoid DC (pDC) and conventional DC (cDC) subsets; the latter is further separated into type 1 (cDC1) and type 2 (cDC2). These cells play a role in initiating and modulating immune responses. Their development is controlled by a network of transcription factors including E proteins consisting of E12, E47, HEB and E2-2, which share a basic helix-loop-helix domain mediating dimerization and DNA binding. Their activity is regulated by their antagonists, Id1-4. The balance between E and Id proteins regulates the transcription of E protein target genes. While E2-2 is essential for pDC differentiation and Id2 is indispensable for cDC1 formation, little is known about how the balance between E and Id proteins controls the early steps of the commitment of common DC precursors (CDP) to the diverse DC subsets. To address this, we used a mouse model that inducibly expresses a chimeric protein, ET2, which contains the DNA binding domain of Tal1 and the transactivation domains of E47. It can compete with Id proteins to bind endogenous E proteins and restore their DNA binding activity, thus increasing net E protein activity. Our data indicated that ET2 expression inhibited the differentiation of pDC and cDC2 subsets in vivo and in vitro. In contrast, cDC1 production was slightly elevated in steady state but dramatically increased in allergic reactions. Furthermore, we detected an augmented proportion of the newly identified cDC1 precursors and decreased representation of cDC2 precursors, suggesting E proteins skew CDPs toward the cDC1 lineage. These results are consistent with reports that E proteins activate IRF8 gene transcription. Further investigation is in progress to elucidate the molecular mechanisms.

Innate lymphoid cells (ILCs) have gained recognition as important regulators in various immune responses. Current paradigm states that all ILCs originate from common lymphoid progenitors (CLP) in the bone marrow through an array of downstream ILC progenitors. Id2, a member of the Id family that inhibits E protein transcription factors, is expressed in these ILC progenitors and is indispensable for ILC differentiation. Interestingly, we found that down-regulating E protein function in the thymus by Id1 overexpression or deletion of two E protein genes resulted in massive expansion of ILC2s in the thymus as well as other organs where ILC2 normally reside. The phenotypic thymic ILC2s expressed ILC2 signature genes and down-regulated T lineage-specific genes. Moreover, Id1 transgenic mice exhibited spontaneous infiltration of eosinophils and heightened type 2 immune responses to the allergen, papain, in the lung. Furthermore, these mice had increased ability in the expulsion of helminth parasites, N. Brasiliensis. These results raise an interesting question as to whether the thymus is a natural site for ILC2 production and the phenotypes of the mutant mice simply highlight such capacity. Indeed, we found that wild type DN1 and DN3 thymocytes can differentiate into IL-5 and IL-13-producing ILC2 cells in vitro. Ablation of E proteins in DN3 cells accelerated the differentiation while gain of E protein function inhibited the process. Together, our data reveal a new ILC2 differentiation program occurring in the thymus and a critical role of E proteins in restraining such a differentiation potential while ensuring proper T cell production.

iNKT cells are a distinct subset of αβ T cells with diverse immunoregulatory functions. Like conventional T cells, iNKT cells arise in thymus from a common CD4+CD8+ double positive (DP) precursor, but these cells are positively selected by glycolipid-loaded CD1d. Our previous result showed that the RAS/MAPK cascade (RAS-Erk-Egr-ID3), a central player during conventional T cell positive selection, is also required for iNKT cell development. Since RAS/MAPK signaling cascade has been reported to promote T cell positive selection by antagonizing E2A activity, we hypothesize that the same mechanism might be applied to iNKT cell development. To test this hypothesis, we utilized a conditional knock-in mouse model expressing ET-2, a chimeric protein composed of the TAD domain of E47 and the bHLH domain of SCL/Tal. Functioning as a dominant negative mutant of ID, ET-2 dramatically impaired iNKT cell development and function. iNKT cells in ET-2 mice were blocked during transition of stage 2 to stage 3, and these cells were also defective in IFN-γ production. Further mechanistic dissection showed that ET-2 might block iNKT cell development through inhibition of T-bet, a transcriptional regulator required for iNKT cell terminal maturation and homeostasis by controlling CD122. Forced expression of T-bet rescued the defects in CD122 expression and IFN-γ production. These results further confirm the role of the RAS/MAPK cascade in iNKT cell development and function.

Complete inhibition of E protein transcription factors by Id1 blocks the developmental transition of CD4/CD8 double negative 1 (DN1, CD44+CD25-) thymocytes to the DN2 (CD44+CD25+) stage. To understand the underlying mechanisms, we observed that mRNA levels of Deltex1, as well as Deltex4, were elevated in Id1-expressing thymocytes, which could result in developmental arrest by attenuating Notch function. In support of this hypothesis, we found that Deltex1 ablation enabled Id1-expressing progenitors to differentiate to the DN3 (CD44-CD25+) stage, which was accompanied by enhanced Notch1 expression in T cell progenitors. Consistently, constitutive activation of Notch1 drove the differentiation of Id1-expressing progenitors to the DN3 stage. Furthermore, we showed that Gfi1b levels decreased whereas GATA3 levels increased in Id1 transgenic thymocytes, and GATA3 expression up-regulated Deltex1 transcription. Thus, T cell commitment may be controlled by the interplay among E proteins, Gfi1b and GATA3 transcription regulators, which influence Notch function through Deltex1 expression. This work was supported by grant from the Nation Institute of Health to X.-H. S. (CA77553).