Viral PB1-F2 and host IFN-γ guide ILC2 and T cell activity during influenza virus infection.

Functional plasticity of innate lymphoid cells (ILCs) and T cells is regulated by host environmental cues, but the influence of pathogen-derived virulence factors has not been described. We now report the interplay between host interferon (IFN)-γ and viral PB1-F2 virulence protein in regulating the functions of ILC2s and T cells that lead to recovery from influenza virus infection of mice. In the absence of IFN-γ, lung ILC2s from mice challenged with the A/California/04/2009 (CA04) H1N1 virus, containing nonfunctional viral PB1-F2, initiated a robust IL-5 response, which also led to improved tissue integrity and increased survival. Conversely, challenge with Puerto Rico/8/1934 (PR8) H1N1 virus expressing fully functional PB1-F2, suppressed IL-5+ ILC2 responses, and induced a dominant IL-13+ CD8 T cell response, regardless of host IFN-γ expression. IFN-γ-deficient mice had increased survival and improved tissue integrity following challenge with lethal doses of CA04, but not PR8 virus, and increased resistance was dependent on the presence of IFN-γR+ ILC2s. Reverse-engineered influenza viruses differing in functional PB1-F2 activity induced ILC2 and T cell phenotypes similar to the PB1-F2 donor strains, demonstrating the potent role of viral PB1-F2 in host resistance. These results show the ability of a pathogen virulence factor together with host IFN-γ to regulate protective pulmonary immunity during influenza infection.

Transcription Factor Bcl11b Controls Identity and Function of Mature Type 2 Innate Lymphoid Cells.

Type 2 innate lymphoid cells (ILC2s) promote anti-helminth responses and contribute to allergies. Here, we report that Bcl11b, previously considered a T-cell-specific transcription factor, acted directly upstream of the key ILC2 transcription factor Gfi1 to maintain its expression in mature ILC2s. Consequently, Bcl11b(-/-) ILC2s downregulated Gata3 and downstream genes, including Il1rl1 (encoding IL-33 receptor), and upregulated Rorc and type 3 ILC (ILC3) genes. Additionally, independent of Gfi1, Bcl11b directly repressed expression of the gene encoding the ILC3 transcription factor Ahr, further contributing to silencing of ILC3 genes in ILC2s. Thus, Bcl11b(-/-) ILC2s lost their functions and gained ILC3 functions, and although they expanded in response to the protease allergen papain, they produced ILC3 but not ILC2 cytokines and caused increased airway infiltration of neutrophils instead of eosinophils. Our results demonstrate that Bcl11b is more than just a T-cell-only transcription factor and establish that Bcl11b sustains mature ILC2 genetic and functional programs and lineage fidelity.

Sequential targeting of interferon pathways for increased host resistance to bacterial superinfection during influenza.

Bacterial co-infections represent a major clinical complication of influenza. Host-derived interferon (IFN) increases susceptibility to bacterial infections following influenza, but the relative roles of type-I versus type-II IFN remain poorly understood. We have used novel mouse models of co-infection in which colonizing pneumococci were inoculated into the upper respiratory tract; subsequent sublethal influenza virus infection caused the bacteria to enter the lungs and mediate lethal disease. Compared to wild-type mice or mice deficient in only one pathway, mice lacking both IFN pathways demonstrated the least amount of lung tissue damage and mortality following pneumococcal-influenza virus superinfection. Therapeutic neutralization of both type-I and type-II IFN pathways similarly provided optimal protection to co-infected wild-type mice. The most effective treatment regimen was staggered neutralization of the type-I IFN pathway early during co-infection combined with later neutralization of type-II IFN, which was consistent with the expression and reported activities of these IFNs during superinfection. These results are the first to directly compare the activities of type-I and type-II IFN during superinfection and provide new insights into potential host-directed targets for treatment of secondary bacterial infections during influenza.

The Interleukin-33-Group 2 Innate Lymphoid Cell Axis Represents a Potential Adjuvant Target To Increase the Cross-Protective Efficacy of Influenza Vaccine.

Interleukin-33 (IL-33) is a multifunctional cytokine that mediates type 2-dominated immune responses. In contrast, the role of IL-33 during viral vaccination, which often aims to induce type 1 immunity, has not been fully investigated. Here, we examined the effects of IL-33 on influenza vaccine responses. We found that intranasal coadministration of IL-33 with an inactivated influenza virus vaccine increases vaccine efficacy against influenza virus infection, not only with the homologous strain but also with heterologous strains, including the 2009 H1N1 influenza virus pandemic strain. Cross-protection was dependent on group 2 innate lymphoid cells (ILC2s), as the beneficial effect of IL-33 on vaccine efficacy was abrogated in ILC2-deficient C57BL/6 Il7rCre/+ Rorafl/fl mice. Furthermore, mechanistic studies revealed that IL-33-activated ILC2s potentiate vaccine efficacy by enhancing mucosal humoral immunity, particularly IgA responses, potentially in a Th2 cytokine-dependent manner. Our results demonstrate that IL-33-mediated activation of ILC2s is a critical early event that is important for the induction of mucosal humoral immunity, which in turn is responsible for cross-strain protection against influenza. Thus, we reveal a previously unrecognized role for the IL-33-ILC2 axis in establishing broadly protective and long-lasting humoral mucosal immunity against influenza, knowledge that may help in the development of a universal influenza vaccine. IMPORTANCE Current influenza vaccines, although capable of protecting against predicted viruses/strains included in the vaccine, are inept at providing cross-protection against emerging/novel strains. Thus, we are in critical need of a universal vaccine that can protect against a wide range of influenza viruses. Our novel findings show that a mucosal vaccination strategy involving the activation of lung ILC2s is highly effective in eliciting cross-protective humoral immunity in the lungs. This suggests that the biology of lung ILC2s can be exploited to increase the cross-reactivity of commercially available influenza subunit vaccines.

Effects of Influenza on Alveolar Macrophage Viability Are Dependent on Mouse Genetic Strain.

Secondary bacterial coinfections following influenza virus pose a serious threat to human health. Therefore, it is of significant clinical relevance to understand the immunological causes of this increased susceptibility. Influenza-induced alterations in alveolar macrophages (AMs) have been shown to be a major underlying cause of the increased susceptibility to bacterial superinfection. However, the mechanisms responsible for this remain under debate, specifically in terms of whether AMs are depleted in response to influenza infection or are maintained postinfection, but with disrupted phagocytic activity. The data presented in this article resolves this issue by showing that either mechanism can differentially occur in individual mouse strains. BALB/c mice exhibited a dramatic IFN-γ-dependent reduction in levels of AMs following infection with influenza A, whereas AM levels in C57BL/6 mice were maintained throughout the course of influenza infection, although the cells displayed an altered phenotype, namely an upregulation in CD11b expression. These strain differences were observed regardless of whether infection was performed with low or high doses of influenza virus. Furthermore, infection with either the H1N1 A/California/04/2009 (CA04) or H1N1 A/PR8/1934 (PR8) virus strain yielded similar results. Regardless of AM viability, both BALB/c and C57BL/6 mice showed a high level of susceptibility to postinfluenza bacterial infection. These findings resolve the apparent inconsistencies in the literature, identify mouse strain-dependent differences in the AM response to influenza infection, and ultimately may facilitate translation of the mouse model to clinical application.

Transcription factor Bcl11b sustains iNKT1 and iNKT2 cell programs, restricts iNKT17 cell program, and governs iNKT cell survival.

Invariant natural killer T (iNKT) cells are innate-like T cells that recognize glycolipid antigens and play critical roles in regulation of immune responses. Based on expression of the transcription factors (TFs) Tbet, Plzf, and Rorγt, iNKT cells have been classified in effector subsets that emerge in the thymus, namely, iNKT1, iNKT2, and iNKT17. Deficiency in the TF Bcl11b in double-positive (DP) thymocytes has been shown to cause absence of iNKT cells in the thymus and periphery due to defective self glycolipid processing and presentation by DP thymocytes and undefined intrinsic alterations in iNKT precursors. We used a model of cre-mediated postselection deletion of Bcl11b in iNKT cells to determine its intrinsic role in these cells. We found that Bcl11b is expressed equivalently in all three effector iNKT subsets, and its removal caused a reduction in the numbers of iNKT1 and iNKT2 cells, but not in the numbers of iNKT17 cells. Additionally, we show that Bcl11b sustains subset-specific cytokine production by iNKT1 and iNKT2 cells and restricts expression of iNKT17 genes in iNKT1 and iNKT2 subsets, overall restraining the iNKT17 program in iNKT cells. The total numbers of iNKT cells were reduced in the absence of Bcl11b both in the thymus and periphery, associated with the decrease in iNKT1 and iNKT2 cell numbers and decrease in survival, related to changes in survival/apoptosis genes. Thus, these results extend our understanding of the role of Bcl11b in iNKT cells beyond their selection and demonstrate that Bcl11b is a key regulator of iNKT effector subsets, their function, identity, and survival.

The multifaceted roles of Bcl11b in thymic and peripheral T cells: impact on immune diseases.

The transcription factor Bcl11b is expressed in all T cell subsets and progenitors, starting from the DN2 stage of T cell development, and it regulates critical processes implicated in the development, function, and survival of many of these cells. Among the common roles of Bcl11b in T cell progenitors and mature T cell subsets are the repression of the innate genetic program and, to some extent, expression maintenance of TCR-signaling components. However, Bcl11b also has unique roles in specific T cell populations, suggesting that its functions depend on cell type and activation state of the cell. In this article, we provide a comprehensive review of the roles of Bcl11b in progenitors, effector T cells, regulatory T cells, and invariant NKT cells, as well as its impact on immune diseases. While emphasizing common themes, including some that might be extended to skin and neurons, we also describe the control of specific functions in different T cell subsets.

Transcription factor Bcl11b controls selection of invariant natural killer T-cells by regulating glycolipid presentation in double-positive thymocytes.

Invariant natural killer T cells (iNKT cells) are innate-like T cells important in immune regulation, antimicrobial protection, and anti-tumor responses. They express semi-invariant T cell receptors, which recognize glycolipid antigens. Their positive selection is mediated by double-positive (DP) thymocytes, which present glycolipid self-antigens through the noncanonical MHC class I-like molecule CD1d. Here we provide genetic and biochemical evidence that removal of the transcription factor Bcl11b in DP thymocytes leads to an early block in iNKT cell development, caused by both iNKT cell extrinsic and intrinsic defects. Specifically, Bcl11b-deficient DP thymocytes failed to support Bcl11b-sufficient iNKT precursor development due to defective glycolipid self-antigen presentation, and showed enlarged lysosomes and accumulation of glycosphingolipids. Expression of genes encoding lysosomal proteins with roles in sphingolipid metabolism and glycolipid presentation was found to be altered in Bcl11b-deficient DP thymocytes. These include cathepsins and Niemann-Pick disease type A, B, and C genes. Thus, Bcl11b plays a central role in presentation of glycolipid self-antigens by DP thymocytes, and regulates directly or indirectly expression of lysosomal genes, exerting a critical extrinsic role in development of iNKT lineage, in addition to the intrinsic role in iNKT precursors. These studies demonstrate a unique and previously undescribed role of Bcl11b in DP thymocytes, in addition to the critical function in positive selection of conventional CD4 and CD8 single-positive thymocytes.

BCL11B enhances TCR/CD28-triggered NF-kappaB activation through up-regulation of Cot kinase gene expression in T-lymphocytes.

BCL11B is a transcriptional regulator with an important role in T-cell development and leukaemogenesis. We demonstrated recently that BCL11B controls expression from the IL (interleukin)-2 promoter through direct binding to the US1 (upstream site 1). In the present study, we provide evidence that BCL11B also participates in the activation of IL-2 gene expression by enhancing NF-kappaB (nuclear factor kappaB) activity in the context of TCR (T-cell receptor)/CD28-triggered T-cell activation. Enhanced NF-kappaB activation is not a consequence of BCL11B binding to the NF-kappaB response elements or association with the NF-kappaB-DNA complexes, but rather the result of higher translocation of NF-kappaB to the nucleus caused by enhanced degradation of IkappaB (inhibitor of NF-kappaB). The enhanced IkappaB degradation in cells with increased levels of BCL11B was specific for T-cells activated through the TCR, but not for cells activated through TNFalpha (tumour necrosis factor alpha) or UV light, and was caused by increased activity of IkappaB kinase, as indicated by its increase in phosphorylation. As BCL11B is a transcription factor, we investigated whether the expression of genes upstream of IkappaB kinase in the TCR/CD28 signalling pathway was affected by increased BCL11B expression, and found that Cot (cancer Osaka thyroid oncogene) kinase mRNA levels were elevated. Cot kinase is known to promote enhanced IkappaB kinase activity, which results in the phosphorylation and degradation of IkappaB and activation of NF-kappaB. The implied involvement of Cot kinase in BCL11B-mediated NF-kappaB activation in response to TCR activation is supported by the fact that a Cot kinase dominant-negative mutant or Cot kinase siRNA (small interfering RNA) knockdown blocked BCL11B-mediated NF-kappaB activation. In support of our observations, in the present study we report that BCL11B enhances the expression of several other NF-kappaB target genes, in addition to IL-2. In addition, we provide evidence that BCL11B associates with intron 2 of the Cot kinase gene to regulate its expression.

Hectd3 promotes pathogenic Th17 lineage through Stat3 activation and Malt1 signaling in neuroinflammation.

Polyubiquitination promotes proteasomal degradation, or signaling and localization, of targeted proteins. Here we show that the E3 ubiquitin ligase Hectd3 is necessary for pathogenic Th17 cell generation in experimental autoimmune encephalomyelitis (EAE), a mouse model for human multiple sclerosis. Hectd3-deficient mice have lower EAE severity, reduced Th17 program and inefficient Th17 cell differentiation. However, Stat3, but not RORγt, has decreased polyubiquitination, as well as diminished tyrosine-705 activating phosphorylation. Additionally, non-degradative polyubiquitination of Malt1, critical for NF-κB activation and Th17 cell function, is reduced. Mechanistically, Hectd3 promotes K27-linked and K29-linked polyubiquitin chains on Malt1, and K27-linked polyubiquitin chains on Stat3. Moreover, Stat3 K180 and Malt1 K648 are targeted by Hectd3 for non-degradative polyubiquitination to mediate robust generation of RORγt+IL-17Ahi effector CD4+ T cells. Thus, our studies delineate a mechanism connecting signaling related polyubiquitination of Malt1 and Stat3, leading to NF-kB activation and RORγt expression, to pathogenic Th17 cell function in EAE.

Diverting T helper cell trafficking through increased plasticity attenuates autoimmune encephalomyelitis.

Naive T helper cells differentiate into functionally distinct effector subsets that drive specialized immune responses. Recent studies indicate that some of the effector subsets have plasticity. Here, we used an EAE model and found that Th17 cells deficient in the transcription factor BCL11B upregulated the Th2-associated proteins GATA3 and IL-4 without decreasing RAR-related orphan receptor γ (RORγt), IL-17, and GM-CSF levels. Surprisingly, abnormal IL-4 production affected Th17 cell trafficking, diverting migration from the draining lymph nodes/CNS route to the mesenteric lymph nodes/gut route, which ameliorated EAE without overt colitis. T helper cell rerouting in EAE was dependent on IL-4, which enhanced retinoic acid (RA) production by dendritic cells, which further induced expression of gut-homing receptors CCR9 and α4ß7 on Bcl11b-deficient CD4+ T cells. Furthermore, IL-4 treatment or Th2 immunization of wild-type mice with EAE caused no alteration in Th17 cytokines or RORγt, but diverted T helper cell trafficking to the gut, which improved EAE outcome without overt colitis. Our data demonstrate that Th17 cells are permissive to Th2 gene expression without affecting Th17 gene expression. This Th17 plasticity has an impact on trafficking, which is a critical component of the immune response and may represent a possible avenue for treating multiple sclerosis.

Critical role of Bcl11b in suppressor function of T regulatory cells and prevention of inflammatory bowel disease.

Dysregulated CD4(+) T cell responses and alterations in T regulatory cells (T(reg) cells) play a critical role in autoimmune diseases, including inflammatory bowel disease (IBD). The current study demonstrates that removal of Bcl11b at the double-positive stage of T cell development or only in T(reg) cells causes IBD because of proinflammatory cytokine-producing CD4(+) T cells infiltrating the colon. Provision of WT T(reg) cells prevented IBD, demonstrating that alterations in T(reg) cells are responsible for the disease. Furthermore, Bcl11b-deficient T(reg) cells had reduced suppressor activity with altered gene expression profiles, including reduced expression of the genes encoding Foxp3 and IL-10, and up-regulation of genes encoding proinflammatory cytokines. Additionally, the absence of Bcl11b altered the induction of Foxp3 expression and reduced the generation of induced T(reg) cells (iT(reg) cells) after Tgf-ß treatment of conventional CD4(+) T cells. Bcl11b bound to Foxp3 and IL-10 promoters, as well as to critical conserved noncoding sequences within the Foxp3 and IL-10 loci, and mutating the Bcl11b binding site in the Foxp3 promoter reduced expression of a luciferase reporter gene. These experiments demonstrate that Bcl11b is indispensable for T(reg) suppressor function and for maintenance of optimal Foxp3 and IL-10 gene expression, as well as for the induction of Foxp3 expression in conventional CD4(+) T cells in response to Tgf-ß and generation of iT(reg) cells.

Flow cytometry analysis of transcription factors in T lymphocytes.

Detection of transcription factors in immune cell populations, particularly in subpopulations that are represented at low frequencies in lymphoid and nonlymphoid organs, presents a particular challenge when using traditional methods such as western blot analysis. Therefore, development of flow cytometry-based methods which allow identification of transcription factors in specific immune cell populations is of main interest. Here we developed and optimized a methodology for rapid and convenient detection of the transcription factor BCL11B in T lymphocyte subpopulations using flow cytometry. The optimal protocol employs saponin and Tween 20 both during the fixation and permeabilization steps, and we demonstrate that it is efficient for three anti-BCL11B antibodies covering distinctive BCL11B epitopes. In addition, we prove that the method preserves the staining of surface markers.

Antigen-specific clonal expansion and cytolytic effector function of CD8+ T lymphocytes depend on the transcription factor Bcl11b.

CD8(+) T lymphocytes mediate the immune response to viruses, intracellular bacteria, protozoan parasites, and tumors. We provide evidence that the transcription factor Bcl11b/Ctip2 controls hallmark features of CD8(+) T cell immunity, specifically antigen (Ag)-dependent clonal expansion and cytolytic activity. The reduced clonal expansion in the absence of Bcl11b was caused by altered proliferation during the expansion phase, with survival remaining unaffected. Two genes with critical roles in TCR signaling were deregulated in Bcl11b-deficient CD8(+) T cells, CD8 coreceptor and Plcgamma1, both of which may contribute to the impaired responsiveness. Bcl11b was found to bind the E8I, E8IV, and E8V, but not E8II or E8III, enhancers. Thus, Bcl11b is one of the transcription factors implicated in the maintenance of optimal CD8 coreceptor expression in peripheral CD8(+) T cells through association with specific enhancers. Short-lived Klrg1(hi)CD127(lo) effector CD8(+) T cells were formed during the course of infection in the absence of Bcl11b, albeit in smaller numbers, and their Ag-specific cytolytic activity on a per-cell basis was altered, which was associated with reduced granzyme B and perforin.