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Background Acute graft-versus-host disease (aGVHD) is one of the most common causes of morbidity for patients undergoing allogeneic stem cell transplantation. There is preliminary evidence that activated Group 2 innate lymphoid cells (ILC2s) from wild type (WT) mice reduces the lethality of aGVHD and is effective in treating lower gastrointestinal (GI) tract manifestations of aGVHD. This raises the prospect that ILC2s may be used for cell-based therapy of aGVHD but vigorous investigation is necessary to assess their impacts on different aspects of aGVHD. Genetically engineered mice which either express Id1 protein (Id1tg/tg), an inhibitor of E protein transcription factors or have E protein genes knocked out (dKO) in the thymus produce massive numbers of ILC2s, thus allowing extensive evaluation of ILC2s. We investigated whether these ILC2s have protective effects in aGVHD as WT ILC2s do using an established mouse model of aGVHD. Results bone marrow transplant was performed by irradiating BALB/c strain of recipient mice and transplanting with bone marrow and T cells from the MHC-disparate C57BL/6 strain. We isolated ILC2s from Id1tg/tg and dKO mice and co-transplanted them to study their effects. Our results confirm that activated ILC2s have a protective role in aGVHD, but the effects varied depending on the origin of ILC2s. Co-transplantation of ILC2s from Id1tg/tg mice were beneficial in aGVHD and are especially helpful in ameliorating the skin manifestations of aGVHD. However, ILC2s from dKO mice were less effective at the protection and behaved differently depending on if the cells were isolated from dKO mice were pre-treated with IL-25 in vivo. Conclusion These findings support the notion that thymus-derived ILC2s from Id1tg/tg mice are protective against aGVHD, with a significant improvement of skin lesions and they behave differently from dKO mice in the setting of aGVHD.

The thymus provides a microenvironment conducive to the differentiation of innate lymphoid cells (ILCs), supplying IL-7 as well as Notch ligands. Early T cell precursors also express a number of obligatory transcription factors essential for ILC differentiation. Therefore, the thymus could be a powerhouse for ILC production. However, coordinated regulation by transcription factors and T cell receptor signaling events ensure that T cell production is the dominating output of the thymus. One group of the key regulators are the basic helix-loop-helix E protein transcription factors and their inhibitors, Id proteins. When E protein activities are downregulated, T cell development is blocked and massive ILC2 production occurs in the thymus. Normally, the thymus indeed generates a small number of ILCs, mostly group 2 ILCs (ILC2s). It has been shown in vitro that ILC2s can be differentiated from multipotent early T cell progenitors (ETPs) as well as committed T cell precursors. Moreover, thymus-derived ILC precursors have been found in the blood of adult mice. They then home to peripheral tissues and undergo differentiation into distinct ILC subsets. These ILC precursors may replenish tissue ILC pools in steady state or on demand in pathophysiological conditions. Collectively, emerging evidence suggests that the thymus plays an underappreciated role in ILC homeostasis.

Qian et al. shows that ILC2s can be generated from not only thymic multipotent progenitors but also committed T cell precursors. These processes are greatly suppressed by E protein transcription factors. Thymic ILC2s show functional differences from those made elsewhere., Current models propose that group 2 innate lymphoid cells (ILC2s) are generated in the bone marrow. Here, we demonstrate that subsets of these cells can differentiate from multipotent progenitors and committed T cell precursors in the thymus, both in vivo and in vitro. These thymic ILC2s exit the thymus, circulate in the blood, and home to peripheral tissues. Ablation of E protein transcription factors greatly promotes the ILC fate while impairing B and T cell development. Consistently, a transcriptional network centered on the ZBTB16 transcription factor and IL-4 signaling pathway is highly up-regulated due to E protein deficiency. Our results show that ILC2 can still arise from what are normally considered to be committed T cell precursors, and that this alternative cell fate is restrained by high levels of E protein activity in these cells. Thymus-derived lung ILC2s of E protein–deficient mice show different transcriptomes, proliferative properties, and cytokine responses from wild-type counterparts, suggesting potentially distinct functions., Graphical Abstract

Dendritic cell (DC) specification and differentiation are controlled by a circuit of transcription factors, which regulate the expression of DC effector genes as well as the transcription factors themselves. E proteins are a widely expressed basic helix-loop-helix family of transcription factors whose activity is suppressed by their inhibitors, ID proteins. Loss-of-function studies have demonstrated the essential role of both E and ID proteins in different aspects of DC development. In this study, we employed a gain-of-function approach to illustrate the importance of the temporal control of E protein function in maintaining balanced differentiation of conventional DC (cDC) subsets, cDC1 and cDC2. We expressed an E protein mutant, ET2, which dimerizes with endogenous E proteins to overcome inhibition by ID proteins and activate the transcription of E protein targets. Induction of ET2 expression at the hematopoietic progenitor stage led to a dramatic reduction in cDC2 precursors (pre-cDC2s) with little impact on pre-cDC1s. Consequently, we observed decreased numbers of cDC2s in the spleen and lung, as well as in FLT3L-driven bone marrow-derived DC cultures. Furthermore, in mice bearing ET2, we detected increased expression of the IRF8 transcription factor in cDC2s, in which IRF8 is normally down-regulated and IRF4 up-regulated. This aberrant expression of IRF8 induced by ET2 may contribute to the impairment of cDC2 differentiation. In addition, analyses of the transcriptomes of splenic cDC1s and cDC2s revealed that ET2 expression led to a shift, at least in part, of the transcriptional profile characteristic of cDC2s to that of cDC1. Together, these results suggest that a precise control of E protein activity is crucial for balanced DC differentiation.

Background Innate lymphoid cells (ILCs) are a subset of innate immune cells that mimic the functions of T lymphocytes, but lack rearranged antigen receptors or clonal expansion in response to antigens. They are characterized in human peripheral blood as Lin− (CD3, CD1a, CD4, CD14, CD16, CD19, CD34, CD94, CD303, FceRI) CD127+ cells. ILCs are divided into three sub-groups: ILC type 1, 2 and 3. The recovery of these cells after allogeneic hematopoietic stem cell transplant (aHSCT) in the pediatric population has not been described. Objectives The primary objective is to study the recovery of ILCs after aHSCT in pediatric patients. The secondary objective was to determine if recovery of ILCs correlated with the incidence or severity of graft-versus-host disease (GVHD). Methods We are conducting a prospective study, enrolling consecutive patients undergoing aHSCT at our institution. After approval from local IRB, the study opened in May 2019. Blood samples are drawn from patients prior to the conditioning regimen, and then weekly starting on day 0 until day +30 (+/- 3 days except day 0), then on day +60, day +90 and day +180. All blood samples are analyzed on the same day by flow cytometry along with a healthy control (thawed frozen peripheral blood mononuclear cells [PBMCs]), serving as the positive control. Results All eligible patients have consented to the study. Six have been enrolled through September 2019; with the following demographics: median age is 9 years (range 21 months-18 years); 2 malignant, 4 non-malignant; matched sibling (n = 1), unrelated (n = 4 complete match, n=1 mismatched 9/10). All patients received donor bone marrow. Conditioning regimens were variable depending on the indication for transplant, with fludarabine, busulfan and individualized dose rATG being the most commonly utilized regimen (67%). GVHD prophylaxis for all patients was tacrolimus with mini-methotrexate. We observed substantial drop in number of ILCs in response to conditioning regimen in all patients, with nadir on day +14 that plateaued through day +30, followed by recovery by day +60 (figure 1). The one patient who has day +90 sample had a complete recovery of total number of ILCs. Similar trend was seen in type 2 ILCs (Lin− CD127+ CRTH2+) and Type 3 ILCs (Lin− CD127+ CRTH2-ve CD117+) while type 1 ILCs (Lin− CD127+ CRTH2− CD117−) showed a slower drop, reaching a nadir on day +21 with slow recovery by day +60. Interestingly, patients who had GVHD (n = 3), had a higher number of ILCs compared to patients who did not develop GVHD (n = 3) on day 0 (p = 0.0781, 2way ANOVA) (Figure 2). This pattern was statistically significant in type 1 ILCs between the two groups of patients on day 0 (p = 0.0062, 2way ANOVA) (Figure 3). Conclusion It is feasible to prospectively study the recovery of ILCs in the post aHSCT pediatric population. We plan to enroll additional patients allowing us to better evaluate a possible correlation between ILCs and GVHD.

Dendritic cells (DCs) initiate immune responses in barrier tissues including lung and skin. Conventional DC (cDC) subsets, CD11b− (cDC1s) or CD11b+ (cDC2s), arise via distinct networks of transcription factors involving IFN regulatory factor 4 (IRF4) and IRF8, and are specialized for unique functional responses. Using mice in which a conditional Irf4 or Irf8 allele is deleted in CD11c+ cells, we determined whether IRF4 or IRF8 deficiency beginning in CD11c+ cDC precursors (pre-cDCs) changed the homeostasis of mature DCs or pre-DCs in the lung, dermis, and spleen. CD11c-cre-Irf4−/− mice selectively lacked a lung-resident CD11chiCD11b+SIRPα+CD24+ DC subset, but not other lung CD11b+ DCs or alveolar macrophages. Numbers of CD11b+CD4+ splenic DCs, but not CD11b+ dermal DCs, were reduced, indicating cDC2s in the lung and dermis develop via different pathways. Irf4 deficiency did not alter numbers of cDC1s. CD11c-cre-Irf8−/− mice lacked lung-resident CD103+ DCs and splenic CD8α+ DCs, yet harbored increased IRF4-dependent DCs. This correlated with a reduced number of Irf8−/− pre-cDCs, which contained elevated IRF4, suggesting that Irf8 deficiency diverts pre-cDC fate. Analyses of Irf4 and Irf8 haploinsufficient mice showed that, although one Irf4 allele was sufficient for lung cDC2 development, two functional Irf8 alleles were required for differentiation of lung cDC1s. Thus, IRF8 and IRF4 act in pre-cDCs to direct the terminal differentiation of cDC1 and cDC2 subsets in the lung and spleen. These data suggest that variation in IRF4 or IRF8 levels resulting from genetic polymorphisms or environmental cues will govern tissue DC numbers and, therefore, regulate the magnitude of DC functional responses.

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.

Background Distant metastasis resulting from vascular dissemination of cancer cells is the primary cause of mortality from breast cancer. We have previously reported that E-selectin expression on the endothelial cell surface mediates shear-resistant adhesion and migration of circulating cancer cells via interaction with CD44. As a result of shedding, soluble E-selectin (sE-selectin) from the activated endothelium is present in the serum. In this study, we aimed to understand the role of sE-selectin in tumor progression and metastasis. Methods We investigated the effect of sE-selectin on shear-resistant adhesion and migration of metastatic breast cancer cells and leukocytes in vitro and in vivo. Results We found that sE-selectin promoted migration and shear-resistant adhesion of CD44+/high breast cancer cell lines (MDA-MB-231 and MDA-MB-468) to non-activated human microvessel endothelial cells (ES-HMVECs), but not of CD44-/low breast cancer cell lines (MCF-7 and T-47D). This endothelial E-selectin independent, sE-selectin-mediated shear-resistant adhesion was also observed in a leukocyte cell line (HL-60) as well as human peripheral blood mononuclear cells (PBMCs). Additionally, the incubation of MDA-MB-231 cells with sE-selectin triggered FAK phosphorylation and shear-resistant adhesion of sE-selectin-treated cells resulted in increased endothelial permeabilization. However, CD44 knockdown in MDA-MB-231 and HL-60 cells resulted in a significant reduction of sE-selectin-mediated shear-resistant adhesion to non-activated HMVECs, suggesting the involvement of CD44/FAK. Moreover, functional blockade of ICAM-1 in non-activated HMVECs resulted in a marked reduction of sE-selectin-mediated shear-resistant adhesion. Finally, the pre-incubation of CD44+ 4 T1 murine breast cancer cells with sE-selectin augmented infiltration into the lung in E-selectin K/O mice and infusion of human PBMCs pre-incubated with sE-selectin stimulated MDA-MB-231 xenografted breast tumor growth in NSG mice. Conclusions Our data suggest that circulating sE-selectin stimulates a broad range of circulating cells via CD44 and mediates pleiotropic effects that promote migration and shear-resistant adhesion in an endothelial E-selectin independent fashion, in turn accelerating tissue infiltration of leukocytes and cancer cells. Electronic supplementary material The online version of this article (doi:10.1186/s12885-016-2366-2) contains supplementary material, which is available to authorized users.

Hematogenous metastasis is a primary cause of mortality from metastatic cancer. The shear-resistant adhesion of circulating tumor cells to the vascular endothelial cell surface under blood flow is an essential step in cell extravasation and further tissue invasion. This is similar to a process exploited by leukocytes for adhesion to inflamed blood vessels (leukocyte mimicry). The shear resistant adhesion is mediated by high affinity interactions between endothelial adhesion molecules and their counter receptor ligand expressed on circulating cells. Thus, weak interaction results in a rapid detachment of circulating cells from endothelium. Despite the critical role of vascular adhesion of cancer cells in hematogenous metastasis, our knowledge regarding this process has been limited due to the difficulty of mimicking dynamic flow conditions in vitro. In order to gain better insight into the shear-resistant adhesion of cancer cells to the endothelium, we developed a protocol for measuring the shear resistant adhesion of circulating tumor cells to endothelial cells under physiologic flow conditions by adapting a well established flow adhesion assay for inflammatory cells. This technique is useful to evaluate 1) the shear resistant adhesion competency of cancer cells and 2) the endothelial adhesion molecules necessary to support cancer cell adhesion (Kang et al., 2015).

Type I IFNs are potent regulators of innate and adaptive immunity and are implicated in the pathogenesis of systemic lupus erythematosus. Here we report that clinical and pathological lupus nephritis and serum anti-nuclear Ab levels are greatly attenuated in New Zealand Mixed (NZM) 2328 mice deficient in type I IFN receptors (IFNAR). To determine whether the inflammatory environment in NZM 2328 mice leads to IFNAR-regulated changes in dendritic cells (DC), the number, activation, and function of DC subsets were compared in 2- and 5-mo-old (clinically healthy) female NZM and NZM-IFNAR−/− mice. Numbers of activated CD40high plasmacytoid DC (pDC) were significantly increased in renal lymph nodes of 2-mo-old NZM but not NZM-IFNAR−/− mice, suggesting an early IFNAR-dependent expansion and activation of pDC at disease sites. Relative to NZM spleens, NZM-IFNAR−/− spleens in 5-mo-old mice were significantly decreased in size and contained reduced numbers of conventional DC subsets, but not pDC. Splenic and renal lymph node NZM-IFNAR−/− DC analyzed directly ex vivo expressed significantly less CD40, CD86, and PDL1 than did NZM DC. Upon activation with synthetic TLR9 ligands in vitro, splenic NZM-IFNAR−/− DC produced less IL-12p40/70 and TNF-α than did NZM DC. The limited IFNAR−/− DC response to endogenous activating stimuli correlated with reduced numbers of splenic activated memory CD4+ T cells and CD19+ B cells in older mice. Thus, IFNAR signaling significantly increases DC numbers, acquisition of Ag presentation competence, and proinflammatory function before onset of clinically apparent lupus disease.

In cutaneous and inhalational Bacillus anthracis infection, innate responses by tissue dendritic cells (DC) will direct adaptive immunity. To dissect these responses, human DCs were incubated with killed vegetative bacteria or highly purified cell wall peptidoglycan (PGN) of the B. anthracis ΔSterne strain. Both vegetative bacteria and PGN stimulated the activation of human DCs, which involved upregulation of surface HLA-DR, the T cell costimulatory molecules CD40 and CD86, the lymph node homing chemokine receptor CCR7, and CD83. B. anthracis PGN and vegetative bacteria elicited production of TNFα, IL-6, and IL-23, but not IL-12p70 or IL-10. To determine the effect of this cytokine profile on CD4+ T cell differentiation, we compared the ability of B. anthracis PGN- and Escherichia coli LPS-stimulated DCs to induce the differentiation of naïve CD4+ T cells into T helper (TH) subsets. While LPS-exposed DCs induced the polarization of CD4+ T cells into TH1 cells producing IFNγ, PGN-exposed DCs directed the differentiation of TH17 cells producing IL-17. Exposure of DCs to either PGN or LPS induced TFH cells producing IL-21. In sum, B. anthracis PGN is a highly stimulatory cell wall component that activates human DCs to display T cell costimulatory molecules and secrete IL-6 and IL-23. Thus, in vivo, PGN-exposed DCs are likely to promote the differentiation of proinflammatory TH17 cells important for extracellular bacteria infections and TFH cells important for humoral immunity.

17-β-estradiol at physiological levels regulates GM-CSF-mediated dendritic cell (DC) differentiation in vitro by promoting the generation of CD11bintLy6C- DC from myeloid progenitors. In this model, estrogen receptor alpha (ER) signaling induces and sustains elevated levels of interferon regulatory factor 4 (IRF4), a transcription factor critical in this pathway. Such events may relate to the de novo DC differentiation that occurs in vivo upon inflammation during infection or autoimmunity, resulting in inflammatory DC that determine the induction of specific Th cell populations. The current work evaluates whether ER signaling regulates new DC differentiation from blood monocytes during inflammation in vivo, and if this is associated with elevated IRF4 expression. ERα+/+ or ERα-/- mice were immunized subcutaneously with complete Freund’s adjuvant. By day 2 post immunization, compared to ERα-/- mice, ERα+/+ mice harbored an increased frequency of CD115+CD11b+ blood monocytes expressing higher levels of IRF4 and MHC class II. On days 2 and 7, relative to ERα-/- mice, lymph nodes of ERα+/+ mice contained greater numbers of monocyte-derived Gr1+CD11b+ CD11clo inflammatory DC. These data show that during inflammation in vivo, ER signaling promotes monocyte activation, IRF4 expression and differentiation, which leads to increased numbers of a de novo inflammatory DC population in draining lymph nodes.

CD207/langerin+ dendritic cells in the epidermis (eLC) and dermis (dLC) initiate immune responses in the skin. We previously published that estrogen receptor alpha (ERα) signaling promotes the differentiation of langerin+ DC in a GM-CSF-driven culture model. To compare the efficiency of dLC differentiation from ERα+ and ERα-/- bone marrow (BM) cells in vivo, we generated mixed competitive ERα+/ERα-/- BM chimeric mice. In the radiation-induced inflammatory environment early after reconstitution, dLC in cutaneous lymph nodes (cLN) were derived preferentially from ERα+ BM cells, indicating that during systemic inflammation ERα signaling promotes dLC differentiation from short term repopulating progenitors. To determine the relative ability of ERα+ and ERα-/- LC to become activated during local skin inflammation, green fluorescent dye in acetone/dibutylphthalate was applied to the skin of ERα+ and ERα-/- mice. Compared to ERα-/- mice, after 24 hr, ERα+ mice contained significantly greater numbers of langerin+ CD11b+ eLC that had acquired the green dye and migrated to cLN. Moreover, ERα+ mice contained higher numbers of new inflammatory CD11bhi CD11clo F4/80+ myeloid cells in the spleen. In sum, ERα signaling in response to endogenous estrogens promotes the development, activation and migration of skin-derived langerin+ DC during inflammation in vivo. Supported by OCAST HR06-157 & NIH AI079616, RR020143RR020143.

De novo differentiation of inflammatory dendritic cells (DC) and macrophages (MΦ) occurs in autoimmune diseases that are strongly female biased in incidence. We previously showed that estrogen receptor alpha (ERα) signaling promotes DC differentiation mediated by GM-CSF, an inflammatory pathway. To compare the efficiency of myeloid cell differentiation from ERα+ and ERα-/- bone marrow (BM) cells in vivo, we generated mixed competitive ERα+/ERα-/- BM chimeric mice. In the radiation-induced inflammatory environment 3 weeks post reconstitution, DC and MΦ in spleen and BM were derived preferentially from ERα+ BM cells, and ERα+ DC displayed higher levels of CD86. These data show that during inflammation ERα signaling promotes the new differentiation and activation of myeloid cells. In contrast, in the homeostatic environment 3 months post reconstitution, DC, MΦ and lymphocytes were predominantly derived from ERα-/- BM cells, consistent with reports that ectopic estradiol depletes hematopoietic progenitors. In chimeric mice, ER signaling also increased splenic DC activation induced by in vivo exposure to TLR9 ligands: ERα+ cDC displayed significantly more CD86 than ERα-/- DC. Thus, during inflammation in vivo, ERα signaling in response to physiological levels of endogenous estrogens promotes DC and MΦ development and activation. Supported by OCAST HR06-157 & NIH AI079616, RR020143.

Distant metastasis resulting from vascular dissemination of cancer cells is the primary cause of mortality from breast cancer. We have previously reported that E-selectin expression on the endothelial cell surface mediates shear-resistant adhesion and migration of circulating cancer cells via interaction with CD44. As a result of shedding, soluble E-selectin (sE-selectin) from the activated endothelium is present in the serum. In this study, we aimed to understand the role of sE-selectin in tumor progression and metastasis.We investigated the effect of sE-selectin on shear-resistant adhesion and migration of metastatic breast cancer cells and leukocytes in vitro and in vivo.We found that sE-selectin promoted migration and shear-resistant adhesion of CD44(+) (/high) breast cancer cell lines (MDA-MB-231 and MDA-MB-468) to non-activated human microvessel endothelial cells (ES-HMVECs), but not of CD44(-/low) breast cancer cell lines (MCF-7 and T-47D). This endothelial E-selectin independent, sE-selectin-mediated shear-resistant adhesion was also observed in a leukocyte cell line (HL-60) as well as human peripheral blood mononuclear cells (PBMCs). Additionally, the incubation of MDA-MB-231 cells with sE-selectin triggered FAK phosphorylation and shear-resistant adhesion of sE-selectin-treated cells resulted in increased endothelial permeabilization. However, CD44 knockdown in MDA-MB-231 and HL-60 cells resulted in a significant reduction of sE-selectin-mediated shear-resistant adhesion to non-activated HMVECs, suggesting the involvement of CD44/FAK. Moreover, functional blockade of ICAM-1 in non-activated HMVECs resulted in a marked reduction of sE-selectin-mediated shear-resistant adhesion. Finally, the pre-incubation of CD44(+) 4 T1 murine breast cancer cells with sE-selectin augmented infiltration into the lung in E-selectin K/O mice and infusion of human PBMCs pre-incubated with sE-selectin stimulated MDA-MB-231 xenografted breast tumor growth in NSG mice.Our data suggest that circulating sE-selectin stimulates a broad range of circulating cells via CD44 and mediates pleiotropic effects that promote migration and shear-resistant adhesion in an endothelial E-selectin independent fashion, in turn accelerating tissue infiltration of leukocytes and cancer cells.