Perinatal Activation of the Interleukin-33 Pathway Promotes Type 2 Immunity in the Developing Lung.

Allergic disease originates in early life and polymorphisms in interleukin-33 gene (IL33) and IL1RL1, coding for IL-33R and decoy receptor sST2, confer allergy risk. Early life T helper 2 (Th2) cell skewing and allergy susceptibility are often seen as remnants of feto-maternal symbiosis. Here we report that shortly after birth, innate lymphoid type 2 cells (ILC2s), eosinophils, basophils, and mast cells spontaneously accumulated in developing lungs in an IL-33-dependent manner. During the phase of postnatal lung alveolarization, house dust mite exposure further increased IL-33, which boosted cytokine production in ILC2s and activated CD11b+ dendritic cells (DCs). IL-33 suppressed IL-12p35 and induced OX40L in neonatal DCs, thus promoting Th2 cell skewing. Decoy sST2 had a strong preventive effect on asthma in the neonatal period, less so in adulthood. Thus, enhanced neonatal Th2 cell skewing to inhaled allergens results from postnatal hyperactivity of the IL-33 axis during a period of maximal lung remodeling.

Epigenomic partitioning of a polygenic risk score for asthma reveals distinct genetically driven disease pathways.

BACKGROUND: Individual differences in susceptibility to developing asthma, a heterogeneous chronic inflammatory lung disease, are poorly understood. Whether genetics can predict asthma risk and how genetic variants modulate the complex pathophysiology of asthma are still debated. AIM: To build polygenic risk scores for asthma risk prediction and epigenomically link predictive genetic variants to pathophysiological mechanisms. METHODS: Restricted polygenic risk scores were constructed using single nucleotide variants derived from genome-wide association studies and validated using data generated in the Rotterdam Study, a Dutch prospective cohort of 14 926 individuals. Outcomes used were asthma, childhood-onset asthma, adulthood-onset asthma, eosinophilic asthma and asthma exacerbations. Genome-wide chromatin analysis data from 19 disease-relevant cell types were used for epigenomic polygenic risk score partitioning. RESULTS: The polygenic risk scores obtained predicted asthma and related outcomes, with the strongest associations observed for childhood-onset asthma (2.55 odds ratios per polygenic risk score standard deviation, area under the curve of 0.760). Polygenic risk scores allowed for the classification of individuals into high-risk and low-risk groups. Polygenic risk score partitioning using epigenomic profiles identified five clusters of variants within putative gene regulatory regions linked to specific asthma-relevant cells, genes and biological pathways. CONCLUSIONS: Polygenic risk scores were associated with asthma(-related traits) in a Dutch prospective cohort, with substantially higher predictive power observed for childhood-onset than adult-onset asthma. Importantly, polygenic risk score variants could be epigenomically partitioned into clusters of regulatory variants with different pathophysiological association patterns and effect estimates, which likely represent distinct genetically driven disease pathways. Our findings have potential implications for personalised risk mitigation and treatment strategies.

Bruton's tyrosine kinase inhibition induces rewiring of proximal and distal B-cell receptor signaling in mice.

Bruton's tyrosine kinase (Btk) is a crucial signaling molecule in BCR signaling and a key regulator of B- cell differentiation and function. Btk inhibition has shown impressive clinical efficacy in various B-cell malignancies. However, it remains unknown whether inhibition additionally induces changes in BCR signaling due to feedback mechanisms, a phenomenon referred to as BCR rewiring. In this report, we studied the impact of Btk activity on major components of the BCR signaling pathway in mice. As expected, NF-κB and Akt/S6 signaling was decreased in Btk-deficient B cells. Unexpectedly, phosphorylation of several proximal signaling molecules, including CD79a, Syk, and PI3K, as well as the key Btk-effector PLCγ2 and the more downstream kinase Erk, were significantly increased. This pattern of BCR rewiring was essentially opposite in B cells from transgenic mice overexpressing Btk. Importantly, prolonged Btk inhibitor treatment of WT mice or mice engrafted with leukemic B cells also resulted in increased phosho-CD79a and phospho-PLCγ2 in B cells. Our findings show that Btk enzymatic function determines phosphorylation of proximal and distal BCR signaling molecules in B cells. We conclude that Btk inhibitor treatment results in rewiring of BCR signaling, which may affect both malignant and healthy B cells.

Phosphoflow Protocol for Signaling Studies in Human and Murine B Cell Subpopulations.

BCR signaling, involving phosphorylation of various downstream molecules, including kinases, lipases, and linkers, is crucial for B cell selection, survival, proliferation, and differentiation. Phosphoflow cytometry (phosphoflow) is a single-cell-based technique to measure phosphorylated intracellular proteins, providing a more quantitative read-out than Western blotting. Recent advances in phosphoflow basically allow simultaneous analysis of protein phosphorylation in B cell (sub)populations, without prior cell sorting. However, fixation and permeabilization procedures required for phosphoflow often affect cell surface epitopes or mAb conjugates, precluding the evaluation of the phosphorylation status of signaling proteins across different B cell subpopulations present in a single sample. In this study, we report a versatile phosphoflow protocol allowing extensive staining of B cell subpopulations in human peripheral blood or various anatomical compartments in the mouse, starting from freshly isolated or frozen cell suspensions. Both human and mouse B cell subpopulations showed different basal and BCR stimulation-induced phosphorylation levels of downstream signaling proteins. For example, peritoneal B-1 cells and splenic marginal zone B cells exhibited significantly increased basal (ex vivo) signaling and increased responsiveness to in vitro BCR stimulation compared with peritoneal B-2 cells and splenic follicular B cells, respectively. In addition, whereas stimulation with anti-IgM or anti-Igκ L chain Abs resulted in strong pCD79a and pPLCγ2 signals, IgD stimulation only induced CD79a but not pPLCγ2 phosphorylation. In summary, the protocol is user friendly and quantifies BCR-mediated phosphorylation with high sensitivity at the single-cell level, in combination with extensive staining to identify individual B cell development and differentiation stages.

Overexpression of SH2-Containing Inositol Phosphatase Contributes to Chronic Lymphocytic Leukemia Survival.

Balanced activity of kinases and phosphatases downstream of the BCR is essential for B cell differentiation and function and is disturbed in chronic lymphocytic leukemia (CLL). In this study, we employed IgH.TEµ mice, which spontaneously develop CLL, and stable EMC CLL cell lines derived from these mice to explore the role of phosphatases in CLL. Genome-wide expression profiling comparing IgH.TEµ CLL cells with wild-type splenic B cells identified 96 differentially expressed phosphatase genes, including SH2-containing inositol phosphatase (Ship2). We found that B cell-specific deletion of Ship2, but not of its close homolog Ship1, significantly reduced CLL formation in IgH.TEµ mice. Treatment of EMC cell lines with Ship1/2 small molecule inhibitors resulted in the induction of caspase-dependent apoptosis. Using flow cytometry and Western blot analysis, we observed that blocking Ship1/2 abrogated EMC cell survival by exerting dual effects on the BCR signaling cascade. On one hand, specific Ship1 inhibition enhanced calcium signaling and thereby abrogated an anergic response to BCR stimulation in CLL cells. On the other hand, concomitant Ship1/Ship2 inhibition or specific Ship2 inhibition reduced constitutive activation of the mTORC1/ribosomal protein S6 pathway and downregulated constitutive expression of the antiapoptotic protein Mcl-1, in both EMC cell lines and primary IgH.TEµ CLL cells. Importantly, also in human CLL, we found overexpression of many phosphatases including SHIP2. Inhibition of SHIP1/SHIP2 reduced cellular survival and S6 phosphorylation and enhanced basal calcium levels in human CLL cells. Taken together, we provide evidence that SHIP2 contributes to CLL pathogenesis in mouse and human CLL.

Bacterial lysate add-on therapy to reduce exacerbations in severe asthma: A double-blind placebo-controlled trial.

BACKGROUND: Asthma exacerbations are frequently induced by respiratory tract infections (RTIs). Bacterial lysates have been described to possess immune-modulatory effects and reduce RTIs as well as asthma symptoms in children. However, whether bacterial lysates have similar effects in adult asthma patients is unknown. AIMS: To reduce asthma exacerbations by add-on bacterial lysate therapy in adults with severe asthma and to characterize the clinical and immune-modulatory effects of this treatment. METHODS: Asthma patients (GINA 4) with ≥2 annual exacerbations in the previous year were included. The intervention regimen consisted of OM-85/placebo for 10 consecutive days per month for 6 months during two winter seasons. Primary end-point was the number of severe asthma exacerbations within 18 months. The study was approved by the national and local ethical review board and registered in the Dutch Trial Registry (NL5752). All participants provided written informed consent. RESULTS: Seventy-five participants were included (38 OM-85; 37 placebo). Exacerbation frequencies were not different between the groups after 18 months (incidence rate ratio 1.07, 95%CI [0.68-1.69], p = 0.77). With the use of OM-85, FEV1% increased by 3.81% (p = 0.04) compared with placebo. Nasopharyngeal swabs taken during RTIs detected a virus less frequently in patients using OM-85 compared to placebo (30.5% vs. 48.0%, p = 0.02). In subjects with type 2 inflammation adherent to the protocol (22 OM-85; 20 placebo), a non-statistically significant decrease in exacerbations in the OM-85 group was observed (IRR = 0.71, 95%CI [0.39-1.26], p = 0.25). Immune-modulatory effects included an increase in several plasma cytokines in the OM-85 group, especially IL-10 and interferons. Peripheral blood T- and B cell subtyping, including regulatory T cells, did not show differences between the groups. CONCLUSION: Although OM-85 may have immune-modulatory effects, it did not reduce asthma exacerbations in this heterogeneous severe adult asthma group. Post hoc analysis showed a potential clinical benefit in patients with type 2 inflammation.

T cells and ILC2s are major effector cells in influenza-induced exacerbation of allergic airway inflammation in mice.

Influenza virus infection is an important cause of severe asthma exacerbations, but it remains unclear how a Th1-mediated antiviral response triggers a prototypical Th2 disease. We investigated CD4+ T cells and group 2 innate lymphoid cells (ILC2s) in influenza virus-infected mice. We found that ILC2s accumulated in the lung rapidly after influenza virus infection, but the induction of IL-5 and IL-13 secretion was delayed and concomitant with T cell activation. In an influenza-induced exacerbation of allergic airway inflammation model we noticed an initial reduction of ILC2 numbers and cytokine production in broncho-alveolar lavage compared to chronic house dust mite (HDM)-mediated airway inflammation alone. ILC2s phenotype was characterized by low T1/ST2, ICOS, KLRG1, and CD25 expression, resembling naïve ILC2s. The contribution of ILC2s to type 2 cytokine production in the early stage of the influenza-induced exacerbation was limited. In contrast, T cells showed increased IL-4 and IL-5 production when exposed to both HDM and influenza virus. Upon virus clearance, ILC2s regained an activated T1/ST2high ICOShigh KLRG1high CD25high phenotype paired with cytokine production and were major contributors to the type 2 cytokine milieu. Collectively, our data indicate that both T cells and ILC2s contribute to influenza-induced exacerbation of allergic airway inflammation, but with different kinetics.

Butyrate inhibits human mast cell activation via epigenetic regulation of FcεRI-mediated signaling.

BACKGROUND: Short-chain fatty acids (SCFAs) are fermented dietary components that regulate immune responses, promote colonic health, and suppress mast cell-mediated diseases. However, the effects of SCFAs on human mast cell function, including the underlying mechanisms, remain unclear. Here, we investigated the effects of the SCFAs (acetate, propionate, and butyrate) on mast cell-mediated pathology and human mast cell activation, including the molecular mechanisms involved. METHOD: Precision-cut lung slices (PCLS) of allergen-exposed guinea pigs were used to assess the effects of butyrate on allergic airway contraction. Human and mouse mast cells were co-cultured with SCFAs and assessed for degranulation after IgE- or non-IgE-mediated stimulation. The underlying mechanisms involved were investigated using knockout mice, small molecule inhibitors/agonists, and genomics assays. RESULTS: Butyrate treatment inhibited allergen-induced histamine release and airway contraction in guinea pig PCLS. Propionate and butyrate, but not acetate, inhibited IgE- and non-IgE-mediated human or mouse mast cell degranulation in a concentration-dependent manner. Notably, these effects were independent of the stimulation of SCFA receptors GPR41, GPR43, or PPAR, but instead were associated with inhibition of histone deacetylases. Transcriptome analyses revealed butyrate-induced downregulation of the tyrosine kinases BTK, SYK, and LAT, critical transducers of FcεRI-mediated signals that are essential for mast cell activation. Epigenome analyses indicated that butyrate redistributed global histone acetylation in human mast cells, including significantly decreased acetylation at the BTK, SYK, and LAT promoter regions. CONCLUSION: Known health benefits of SCFAs in allergic disease can, at least in part, be explained by epigenetic suppression of human mast cell activation.

The DNA-binding protein CTCF limits proximal Vκ recombination and restricts κ enhancer interactions to the immunoglobulin κ light chain locus.

Regulation of immunoglobulin (Ig) V(D)J gene rearrangement is dependent on higher-order chromatin organization. Here, we studied the in vivo function of the DNA-binding zinc-finger protein CTCF, which regulates interactions between enhancers and promoters. By conditional deletion of the Ctcf gene in the B cell lineage, we demonstrate that loss of CTCF allowed Ig heavy chain recombination, but pre-B cell proliferation and differentiation was severely impaired. In the absence of CTCF, the Igκ light chain locus showed increased proximal and reduced distal Vκ usage. This was associated with enhanced proximal Vκ and reduced Jκ germline transcription. Chromosome conformation capture experiments demonstrated that CTCF limits interactions of the Igκ enhancers with the proximal V(κ) gene region and prevents inappropriate interactions between these strong enhancers and elements outside the Igκ locus. Thus, although Ig gene recombination can occur in the absence of CTCF, it is a critical factor determining Vκ segment choice for recombination.

Distinct and Overlapping Functions of TEC Kinase and BTK in B Cell Receptor Signaling.

The Tec tyrosine kinase is expressed in many cell types, including hematopoietic cells, and is a member of the Tec kinase family that also includes Btk. Although the role of Btk in B cells has been extensively studied, the role of Tec kinase in B cells remains largely unclear. It was previously shown that Tec kinase has the ability to partly compensate for loss of Btk activity in B cell differentiation, although the underlying mechanism is unknown. In this study, we confirm that Tec kinase is not essential for normal B cell development when Btk is present, but we also found that Tec-deficient mature B cells showed increased activation, proliferation, and survival upon BCR stimulation, even in the presence of Btk. Whereas Tec deficiency did not affect phosphorylation of phospholipase Cγ or Ca2+ influx, it was associated with significantly increased activation of the intracellular Akt/S6 kinase signaling pathway upon BCR and CD40 stimulation. The increased S6 kinase phosphorylation in Tec-deficient B cells was dependent on Btk kinase activity, as ibrutinib treatment restored pS6 to wild-type levels, although Btk protein and phosphorylation levels were comparable to controls. In Tec-deficient mice in vivo, B cell responses to model Ags and humoral immunity upon influenza infection were enhanced. Moreover, aged mice lacking Tec kinase developed a mild autoimmune phenotype. Taken together, these data indicate that in mature B cells, Tec and Btk may compete for activation of the Akt signaling pathway, whereby the activating capacity of Btk is limited by the presence of Tec kinase.

The Notch pathway inhibitor stapled α-helical peptide derived from mastermind-like 1 (SAHM1) abrogates the hallmarks of allergic asthma.

BACKGROUND: The Notch signaling pathway has been implicated in the pathogenesis of allergic airway inflammation. Targeting the active Notch transactivation complex by using the cell-permeable, hydrocarbon-stapled synthetic peptide stapled α-helical peptide derived from mastermind-like 1 (SAHM1) resulted in genome-wide suppression of Notch-activated genes in leukemic cells and other models. However, the efficacy of SAHM1 in allergic asthma models has remained unexplored. OBJECTIVE: We aimed to investigate the therapeutic efficacy of SAHM1 in a house dust mite (HDM)-driven asthma model. METHODS: Topical therapeutic intervention with SAHM1 or a control peptide was performed during sensitization, challenge, or both with HDM in mice. Airway inflammation was assessed by using multicolor flow cytometry, and bronchial hyperreactivity was studied. Additionally, SAHM1 therapy was investigated in mice with established allergic airway inflammation and in a model in which we neutralized IFN-γ during HDM challenge to support the TH2 response and exacerbate asthma. RESULTS: SAHM1 treatment during the challenge phase led to a marked reduction of eosinophil and T cell numbers in bronchoalveolar lavage fluid compared with those in diluent-treated or control peptide-treated mice. Likewise, T-cell cytokine content and bronchial hyperreactivity were reduced. SAHM1 treatment dampened TH2 inflammation during ongoing HDM challenge and enhanced recovery after established asthma. Additionally, in the presence of anti-IFN-γ antibodies, SAHM1 downregulated expression of the key TH2 transcription factor GATA3 and intracellular IL-4 in bronchoalveolar lavage fluid T cells, but expression of the TH17 transcription factor retinoic acid-related orphan receptor γt or intracellular IL-17 was not affected. SAHM1 therapy also reduced serum IgE levels. CONCLUSIONS: Therapeutic intervention of Notch signaling by SAHM1 inhibits allergic airway inflammation in mice and is therefore an interesting new topical treatment opportunity in asthmatic patients.

Epigenome analysis links gene regulatory elements in group 2 innate lymphocytes to asthma susceptibility.

BACKGROUND: Group 2 innate lymphoid cells (ILC2s) are major producers of the cytokines driving allergic asthma, and increased ILC2 numbers have been detected in blood and sputum of asthmatic patients. Asthma susceptibility has a strong genetic component, but the underlying mechanisms and whether asthma genetics affect ILC2 biology remain unclear. OBJECTIVE: We sought to study the ILC2 transcriptome and epigenome during airway inflammation (AI) to couple these to genes and genetic variants associated with asthma pathogenesis. METHODS: Mice harboring a reporter for the key ILC2 transcription factor GATA-3 were subjected to IL-33-driven AI, and ILC2s were isolated from bronchoalveolar lavage fluid and mediastinal lymph nodes. Human ILC2s were purified from peripheral blood and activated in vitro. We used RNA sequencing, genome-wide identification of histone-3 lysine-4 dimethylation-marked chromatin, and computational approaches to study the ILC2 transcriptome and epigenome. RESULTS: Activated ILC2s in mice displayed a tissue-specific gene expression signature that emerged from remarkably similar epigenomes. We identified superenhancers implicated in controlling ILC2 identity and asthma-associated genes. More than 300 asthma-associated genetic polymorphisms identified in genome-wide association studies localized to H3K4Me2+ gene regulatory elements in ILC2s. A refined set of candidate causal asthma-associated variants was uniquely enriched in ILC2, but not TH2 cell, regulatory regions. CONCLUSIONS: ILC2s in AI use a flexible epigenome that couples adaptation to new microenvironments with functional plasticity. Importantly, we reveal strong correlations between gene regulatory mechanisms in ILC2s and the genetic basis of asthma, supporting a pathogenic role for ILC2s in patients with allergic asthma.

The Role of B Cells in Carriage and Clearance of Mycoplasma pneumoniae From the Respiratory Tract of Mice.

Background: Carriage of Mycoplasma pneumoniae (Mp) in the nasopharynx is considered a prerequisite for pulmonary infection. It is interesting to note that Mp carriage is also detected after infection. Although B cells are known to be involved in pulmonary Mp clearance, their role in Mp carriage is unknown. Methods: In this study, we show in a mouse model that Mp persists in the nose after pulmonary infection, similar to humans. Results: Infection of mice enhanced Mp-specific immunoglobulin (Ig) M and IgG levels in serum and bronchoalveolar lavage fluid. However, nasal washes only contained elevated Mp-specific IgA. These differences in Ig compartmentalization correlated with differences in Mp-specific B cell responses between nose- and lung-draining lymphoid tissues. Moreover, transferred Mp-specific serum Igs had no effect on nasal carriage in B cell-deficient µMT mice, whereas this enabled µMT mice to clear pulmonary Mp infection. Conclusions: We report the first evidence that humoral immunity is limited in clearing Mp from the upper respiratory tract.

Enhanced Expression of Bruton's Tyrosine Kinase in B Cells Drives Systemic Autoimmunity by Disrupting T Cell Homeostasis.

Upon BCR stimulation, naive B cells increase protein levels of the key downstream signaling molecule Bruton's tyrosine kinase (BTK). Transgenic CD19-hBtk mice with B cell-specific BTK overexpression show spontaneous germinal center formation, anti-nuclear autoantibodies, and systemic autoimmunity resembling lupus and Sjögren syndrome. However, it remains unknown how T cells are engaged in this pathology. In this study, we found that CD19-hBtk B cells were high in IL-6 and IL-10 and disrupted T cell homeostasis in vivo. CD19-hBtk B cells promoted IFN-γ production by T cells and expression of the immune-checkpoint protein ICOS on T cells and induced follicular Th cell differentiation. Crosses with CD40L-deficient mice revealed that increased IL-6 production and autoimmune pathology in CD19-hBtk mice was dependent on B-T cell interaction, whereas IL-10 production and IgM autoantibody formation were CD40L independent. Surprisingly, in Btk-overexpressing mice, naive B cells manifested increased CD86 expression, which was dependent on CD40L, suggesting that T cells interact with B cells in a very early stage of immune pathology. These findings indicate that increased BTK-mediated signaling in B cells involves a positive-feedback loop that establishes T cell-propagated autoimmune pathology, making BTK an attractive therapeutic target in autoimmune disease.

Notch signaling in T cells is essential for allergic airway inflammation, but expression of the Notch ligands Jagged 1 and Jagged 2 on dendritic cells is dispensable.

BACKGROUND: Allergic asthma is characterized by a TH2 response induced by dendritic cells (DCs) that present inhaled allergen. Although the mechanisms by which they instruct TH2 differentiation are still poorly understood, expression of the Notch ligand Jagged on DCs has been implicated in this process. OBJECTIVE: We sought to establish whether Notch signaling induced by DCs is critical for house dust mite (HDM)-driven allergic airway inflammation (AAI) in vivo. METHODS: The induction of Notch ligand expression on DC subsets by HDM was quantified by using quantitative real-time PCR. We used an HDM-driven asthma mouse model to compare the capacity of Jagged 1 and Jagged 2 single- and double-deficient DCs to induce AAI. In addition, we studied AAI in mice with a T cell-specific deletion of recombination signal-binding protein for immunoglobulin Jκ region (RBPJκ), a downstream effector of Notch signaling. RESULTS: HDM exposure promoted expression of Jagged 1, but not Jagged 2, on DCs. In agreement with published findings, in vitro-differentiated and HDM-pulsed Jagged 1 and Jagged 2 double-deficient DCs lacked the capacity to induce AAI. However, after in vivo intranasal sensitization and challenge with HDM, DC-specific Jagged 1 or Jagged 2 single- or double-deficient mice had eosinophilic airway inflammation and a TH2 cell activation phenotype that was not different from that in control littermates. In contrast, RBPJκ-deficient mice did not experience AAI and airway hyperreactivity. CONCLUSION: Our results show that the Notch signaling pathway in T cells is crucial for the induction of TH2-mediated AAI in an HDM-driven asthma model but that expression of Jagged 1 or Jagged 2 on DCs is not required.

T cells are necessary for ILC2 activation in house dust mite-induced allergic airway inflammation in mice.

Allergic asthma is a chronic inflammation of the airways mediated by an adaptive type 2 immune response. Upon allergen exposure, group 2 innate lymphoid cells (ILC2s) can be rapidly activated and represent an early innate source of IL-5 and IL-13. Here, we used a house dust mite (HDM)-driven asthma mouse model to study the induction of ILC2s in allergic airway inflammation. In BALF, lungs, and lymph nodes, ILC2 activation is critically dependent on prior sensitization with HDM. Importantly, T cells are required for ILC2 induction, whereby T-cell activation precedes ILC2 induction. During HDM-driven allergic airway inflammation the accumulation of ILC2s in BALF is IL-33 independent, although infiltrating ILC2s produce less cytokines in Il33(-/-) mice. Transfer of in vitro polarized OVA-specific OT-II Th2 cells alone or in combination with Th17 cells followed by OVA and HDM challenge is not sufficient to induce ILC2, despite significant eosinophilic inflammation and T-cell activation. In this asthma model, ILC2s are therefore not an early source of Th2 cytokines, but rather contribute to type 2 inflammation in which Th2 cells play a key role. Taken together, ILC2 induction in HDM-mediated allergic airway inflammation in mice critically depends on activation of T cells.

Pre-B cell receptor signaling induces immunoglobulin κ locus accessibility by functional redistribution of enhancer-mediated chromatin interactions.

During B cell development, the precursor B cell receptor (pre-BCR) checkpoint is thought to increase immunoglobulin κ light chain (Igκ) locus accessibility to the V(D)J recombinase. Accordingly, pre-B cells lacking the pre-BCR signaling molecules Btk or Slp65 showed reduced germline V(κ) transcription. To investigate whether pre-BCR signaling modulates V(κ) accessibility through enhancer-mediated Igκ locus topology, we performed chromosome conformation capture and sequencing analyses. These revealed that already in pro-B cells the κ enhancers robustly interact with the ∼3.2 Mb V(κ) region and its flanking sequences. Analyses in wild-type, Btk, and Slp65 single- and double-deficient pre-B cells demonstrated that pre-BCR signaling reduces interactions of both enhancers with Igκ locus flanking sequences and increases interactions of the 3'κ enhancer with V(κ) genes. Remarkably, pre-BCR signaling does not significantly affect interactions between the intronic enhancer and V(κ) genes, which are already robust in pro-B cells. Both enhancers interact most frequently with highly used V(κ) genes, which are often marked by transcription factor E2a. We conclude that the κ enhancers interact with the V(κ) region already in pro-B cells and that pre-BCR signaling induces accessibility through a functional redistribution of long-range chromatin interactions within the V(κ) region, whereby the two enhancers play distinct roles.

Enforced expression of Gata3 in T cells and group 2 innate lymphoid cells increases susceptibility to allergic airway inflammation in mice.

Airway inflammation in allergic asthma reflects a threshold response of the innate immune system, including group 2 innate lymphoid cells (ILC2), followed by an adaptive Th2 cell-mediated response. Transcription factor Gata3 is essential for differentiation of both Th2 cells and ILC2. We investigated the effects of enforced Gata3 expression in T cells and ILC2 on the susceptibility of mice to allergic airway inflammation (AAI). We used CD2-Gata3 transgenic (Tg) mice with enforced Gata3 expression driven by the CD2 promoter, which is active both in T cells and during ILC2 development. CD2-Gata3 Tg mice and wild-type (WT) littermates were analyzed in mild models of AAI without adjuvants. Whereas OVA allergen exposure did not induce inflammation in WT controls, CD2-Gata3 Tg mice showed clear AAI and enhanced levels of IL-5 and IL-13 in bronchoalveolar lavage. Likewise, in house dust mite-driven asthma, CD2-Gata3 Tg mice were significantly more susceptible to AAI than WT littermates, whereby both ILC2 and Th2 cells were important cellular sources of IL-5 and IL-13 in bronchoalveolar lavage and lung tissue. Compared with WT littermates, CD2-Gata3 Tg mice contained increased numbers of ILC2, which expressed high levels of IL-33R and contributed significantly to early production of IL-4, IL-5, and IL-13. CD2-Gata3 Tg mice also had a unique population of IL-33-responsive non-B/non-T lymphoid cells expressing IFN-γ. Enforced Gata3 expression is therefore sufficient to enhance Th2 and ILC2 activity, and leads to increased susceptibility to AAI after mild exposure to inhaled harmless Ags that otherwise induce Ag tolerance.

Essential, dose-dependent role for the transcription factor Gata3 in the development of IL-5+ and IL-13+ type 2 innate lymphoid cells.

Group 2 innate lymphoid cells (ILC2s; also called nuocytes, innate helper cells, or natural helper cells) provide protective immunity during helminth infection and play an important role in influenza-induced and allergic airway hyperreactivity. Whereas the transcription factor GATA binding protein 3 (Gata3) is important for the production of IL-5 and -13 by ILC2s in response to IL-33 or -25 stimulation, it is not known whether Gata3 is required for ILC2 development from hematopoietic stem cells. Here, we show that chimeric mice generated with Gata3-deficient fetal liver hematopoietic stem cells fail to develop systemically dispersed ILC2s. In these chimeric mice, in vivo administration of IL-33 or -25 fails to expand ILC2 numbers or to induce characteristic ILC2-dependent IL-5 or -13 production. Moreover, cell-intrinsic Gata3 expression is required for ILC2 development in vitro and in vivo. Using mutant and transgenic mice in which Gata3 gene copy number is altered, we show that ILC2 generation from common lymphoid progenitors, as well as ILC2 homeostasis and cytokine production, is regulated by Gata3 expression levels in a dose-dependent fashion. Collectively, these results identify Gata3 as a critical early regulator of ILC2 development, thereby extending the paradigm of Gata3-dependent control of type 2 immunity to include both innate and adaptive lymphocytes.

Surrogate light chain expression beyond the pre-B cell stage promotes tolerance in a dose-dependent fashion.

While surrogate light chain (SLC) expression is normally terminated in differentiating pre-B cells, co-expression of SLC and conventional light chains has been reported in a small population of autoreactive peripheral human B cells that accumulate in arthritic joints. Despite this association with autoimmunity the contribution of SLC expressing mature B cells to disease development is still unknown. We studied the pathogenicity of SLC(+) B cells in a panel of mice that transgenically express the SLC components VpreB and λ5 throughout B cell development. Here we report that although VpreB or λ5 expression mildly activated mature B cells, only moderate VpreB expression levels - in the absence of λ5 - enhanced IgG plasma cell formation. However, no autoantibody production was detectable in VpreB or λ5 transgenic mice and VpreB expression could not accelerate autoimmunity. Instead, moderate VpreB expression partially protected mice from induced autoimmune arthritis. In support of a tolerogenic role of SLC-transgenic B cells, we observed that in a dose-dependent manner SLC expression beyond the pre-B cell stage enhanced clonal deletion among immature and transitional B cells and rendered mature B cells anergic. These findings suggest that SLC expression does not propagate autoimmunity, but instead may impose tolerance.