Denisovan, modern human and mouse TNFAIP3 alleles tune A20 phosphorylation and immunity.

Resisting and tolerating microbes are alternative strategies to survive infection, but little is known about the evolutionary mechanisms controlling this balance. Here genomic analyses of anatomically modern humans, extinct Denisovan hominins and mice revealed a TNFAIP3 allelic series with alterations in the encoded immune response inhibitor A20. Each TNFAIP3 allele encoded substitutions at non-catalytic residues of the ubiquitin protease OTU domain that diminished IκB kinase-dependent phosphorylation and activation of A20. Two TNFAIP3 alleles encoding A20 proteins with partial phosphorylation deficits seemed to be beneficial by increasing immunity without causing spontaneous inflammatory disease: A20 T108A;I207L, originating in Denisovans and introgressed in modern humans throughout Oceania, and A20 I325N, from an N-ethyl-N-nitrosourea (ENU)-mutagenized mouse strain. By contrast, a rare human TNFAIP3 allele encoding an A20 protein with 95% loss of phosphorylation, C243Y, caused spontaneous inflammatory disease in humans and mice. Analysis of the partial-phosphorylation A20 I325N allele in mice revealed diminished tolerance of bacterial lipopolysaccharide and poxvirus inoculation as tradeoffs for enhanced immunity.

Tfh cells set the stage for tumor control.

The microbiome has a profound influence on the development and progression of cancer, as well as the response to immunotherapy. In this issue of Immunity, Overacre-Delgoffe et al. (2021) examine the impact of the immunogenic bacteria Helicobacter hepaticus (Hhep) in the immune response to colorectal cancer, providing evidence that T follicular helper (Tfh) cells that specifically recognize Hhep antigens are necessary for the control of tumor growth.

The retroelement Lx9 puts a brake on the immune response to virus infection.

The notion that mobile units of nucleic acid known as transposable elements can operate as genomic controlling elements was put forward over six decades ago1,2. However, it was not until the advancement of genomic sequencing technologies that the abundance and repertoire of transposable elements were revealed, and they are now known to constitute up to two-thirds of mammalian genomes3,4. The presence of DNA regulatory regions including promoters, enhancers and transcription-factor-binding sites within transposable elements5-8 has led to the hypothesis that transposable elements have been co-opted to regulate mammalian gene expression and cell phenotype8-14. Mammalian transposable elements include recent acquisitions and ancient transposable elements that have been maintained in the genome over evolutionary time. The presence of ancient conserved transposable elements correlates positively with the likelihood of a regulatory function, but functional validation remains an essential step to identify transposable element insertions that have a positive effect on fitness. Here we show that CRISPR-Cas9-mediated deletion of a transposable element-namely the LINE-1 retrotransposon Lx9c11-in mice results in an exaggerated and lethal immune response to virus infection. Lx9c11 is critical for the neogenesis of a non-coding RNA (Lx9c11-RegoS) that regulates genes of the Schlafen family, reduces the hyperinflammatory phenotype and rescues lethality in virus-infected Lx9c11-/- mice. These findings provide evidence that a transposable element can control the immune system to favour host survival during virus infection.

Dual Nature of Type I Interferons in SARS-CoV-2-Induced Inflammation.

Coronavirus disease 2019 (COVID-19) is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The ability of our cells to secrete type I interferons (IFN-Is) is essential for the control of virus replication and for effective antiviral immune responses; for this reason, viruses have evolved the means to antagonize IFN-I. Inhibition of IFN-I production is pronounced in SARS-CoV-2 infection, which can impair the adaptive immune response and exacerbate inflammatory disease at late stages of infection. However, therapeutic boosting of IFN-I offers a narrow time window for efficacy and safety. Here, we discuss how limits placed on IFN-I by SARS-CoV-2 shape the immune response and whether this might be countered with therapeutic approaches and vaccine design.

A fine romance: T follicular helper cells and B cells.

T follicular helper (Tfh) cells help B cells to generate affinity-matured antibodies. Three papers in this issue of Immunity (Choi et al., 2011; Kerfoot et al., 2011; Kitano et al., 2011) provide information about the reciprocal relationship between B cells and Tfh cells.

A subset of interleukin-21+ chemokine receptor CCR9+ T helper cells target accessory organs of the digestive system in autoimmunity.

This study describes a CD4+ T helper (Th) cell subset marked by coexpression of the cytokine interleukin 21 (IL-21) and the gut-homing chemokine receptor CCR9. Although CCR9+ Th cells were observed in healthy mice and humans, they were enriched in the inflamed pancreas and salivary glands of NOD mice and in the circulation of Sjögren's syndrome patients. CCR9+ Th cells expressed large amounts of IL-21, inducible T cell costimulator (ICOS), and the transcription factors Bcl6 and Maf, and also supported antibody production from B cells, thereby resembling T follicular B helper (Tfh) cells. However, in contrast to Tfh cells, CCR9+ Th cells displayed limited expression of CXCR5 and the targets of CCR9+ Th cells were CD8+ T cells whose responsiveness to IL-21 was necessary for the development of diabetes. Thus, CCR9+ Th cells are a subset of IL-21-producing T helper cells that influence regional specification of autoimmune diseases that affect accessory organs of the digestive system.

IL-2 Shapes the Survival and Plasticity of IL-17-Producing γδ T Cells.

IL-17-producing γδ T (γδT-17) cells have proved to be an important early source of IL-17 in many inflammatory settings and are emerging as an important participant in protumor immune responses. Considering that their peripheral activation depends largely on innate signals rather than TCR ligation, it is important to understand what mechanisms exist to curb unwanted activation. Expression of the high-affinity IL-2R on γδT-17 cells prompted us to investigate a role for this cytokine. We found γδT-17 cells to be enriched, not depleted, in IL-2-deficient mice. The absence of IL-2 also resulted in higher IL-17 production and the emergence of IL-17+IFN-γ+ double producers. Furthermore, the addition of IL-2 to in vitro cultures of sorted γδT-17 cells was able to moderate IL-17 and affect differentiation into polyfunctional cytokine-producing cells. Interestingly, the Vγ6+ subset was more susceptible to the effects of IL-2 than Vγ4+ γδT-17 cells. We also found that unlike other γδ T cells, γδT-17 cells do not produce IL-2, but express Blimp-1, a known transcriptional repressor of IL-2. Although IL-2 was able to induce robust proliferation of γδT-17 cells, it did not sustain viability, negatively impacting their survival via downregulation of the IL-7R. Taken together, these data indicate that IL-2 can augment the γδT-17 response in favor of short-lived effectors with limited plasticity, particularly in the presence of IL-1ß and IL-23. In this way, IL-2 may act to curtail the innate-like response of γδT-17 cells upon arrival of IL-2-producing adaptive immune cells at the site of inflammation.

GPR65 inhibits experimental autoimmune encephalomyelitis through CD4+ T cell independent mechanisms that include effects on iNKT cells.

The G protein-coupled receptor 65 (GPR65) gene has been genetically associated with several autoimmune diseases, including multiple sclerosis (MS). GPR65 is predominantly expressed in lymphoid organs and is activated by extracellular protons. In this study, we tested whether GPR65 plays a functional role in demyelinating autoimmune disease. Using a murine model of MS, experimental autoimmune encephalomyelitis (EAE), we found that Gpr65-deficient mice develop exacerbated disease. CD4+ helper T cells are key drivers of EAE pathogenesis, however, Gpr65 deficiency in these cells did not contribute to the observed exacerbated disease. Instead, Gpr65 expression levels were found to be highest on invariant natural killer T (iNKT) cells. EAE severity in Gpr65-deficient mice was normalized in the absence of iNKT cells (CD1d-deficient mice), suggesting that GPR65 signals in iNKT cells are important for suppressing autoimmune disease. These findings provide functional support for the genetic association of GPR65 with MS and demonstrate GPR65 signals suppress autoimmune activity in EAE.

Longitudinal Impact of Smoking and Smoking Cessation on Inflammatory Markers of Cardiovascular Disease Risk.

OBJECTIVE: To evaluate longitudinal changes in 6 inflammatory markers that predict cardiovascular disease events among smokers making a quit attempt and to characterize their cross-sectional associations between smoking and smoking heaviness. APPROACH AND RESULTS: In a longitudinal cohort study of contemporary smokers (n=1652), we evaluated (1) independent associations of smoking heaviness markers (exhaled carbon monoxide, cigarettes/d, pack-years) with inflammatory markers (C-reactive protein, D-dimer, fibrinogen, urinary F2 isoprostane:creatinine [F2:Cr] ratio, white blood cell [WBC] count, myeloperoxidase) and (2) the effects of smoking cessation and continued smoking on these inflammatory markers after 1 year, among the 888 smokers who made an aided quit attempt as part of a randomized comparative effectiveness trial or standard care. There were strong, independent associations between smoking heaviness markers and the F2:Cr ratio, WBC, and myeloperoxidase (all Padj<0.001), but not high-sensitivity C-reactive protein, D-dimer, or fibrinogen. Participants were mean (SD) 49.6 years old (11.6), 54% women, 34% non-white, and smoked 16.8 cigarettes/d (8.5) for 27.3 pack-years (18.6). After 1 year, the 344 successful abstainers gained more weight (4.0 [6.0] versus 0.4 [5.7] pounds; P<0.001) and had larger increases in insulin resistance scores (P=0.02) than continuing smokers. Despite these increases, abstainers had significant decreases in F2:Cr ratio (P<0.001) and WBC counts (P<0.001). Changes in other markers were not related to quitting. CONCLUSIONS: Smoking heaviness is associated with increased F2:Cr ratio, myeloperoxidase, and WBC counts. Cessation improves the F2:Cr ratio and WBC counts independent of weight change, suggesting reduced inflammation related to less oxidant stress.

IL-27 Directly Enhances Germinal Center B Cell Activity and Potentiates Lupus in Sanroque Mice.

Germinal centers (GC) give rise to high-affinity and long-lived Abs and are critical in immunity and autoimmunity. IL-27 supports GCs by promoting survival and function of T follicular helper cells. We demonstrate that IL-27 also directly enhances GC B cell function. Exposure of naive human B cells to rIL-27 during in vitro activation enhanced their differentiation into CD20+CD38+CD27lowCD95+CD10+ cells, consistent with the surface marker phenotype of GC B cells. This effect was inhibited by loss-of-function mutations in STAT1 but not STAT3 To extend these findings, we studied the in vivo effects of IL-27 signals to B cells in the GC-driven Roquinsan/san lupus mouse model. Il27ra-/-Roquinsan/san mice exhibited significantly reduced GCs, IgG2a(c)+ autoantibodies, and nephritis. Mixed bone marrow chimeras confirmed that IL-27 acts through B cell- and CD4+ T cell-intrinsic mechanisms to support GCs and alter the production of pathogenic Ig isotypes. To our knowledge, our data provide the first evidence that IL-27 signals directly to B cells promote GCs and support the role of IL-27 in lupus.

A fundamental role for interleukin-21 in the generation of T follicular helper cells.

T cell help to B cells is a fundamental property of adaptive immunity, yet only recently have many of the cellular and molecular mechanisms of T cell help emerged. T follicular helper (Tfh) cells are the CD4(+) T helper cells that provide cognate help to B cells for high-affinity antibody production in germinal centers (GC). Tfh cells produce interleukin-21 (IL-21), and we show that IL-21 was necessary for GC formation. However, the central role of IL-21 in GC formation reflected its effects on Tfh cell generation rather than on B cells. Expression of the inducible costimulator (ICOS) was necessary for optimal production of IL-21, indicative of interplay between these two Tfh cell-expressed molecules. Finally, we demonstrate that IL-21's costimulatory capacity for T helper cell differentiation operated at the level of the T cell receptor signalosome through Vav1, a signaling molecule that controls T cell helper function. This study reveals a previously unappreciated role for Tfh cells in the formation of the GC and isotype switching through a CD4(+) T cell-intrinsic requirement for IL-21.

IL-21 and IL-4 Collaborate To Shape T-Dependent Antibody Responses.

The selection of affinity-matured Ab-producing B cells is supported by interactions with T follicular helper (Tfh) cells. In addition to cell surface-expressed molecules, cytokines produced by Tfh cells, such as IL-21 and IL-4, provide B cell helper signals. In this study, we analyze how the fitness of Th cells can influence Ab responses. To do this, we used a model in which IL-21R-sufficient (wild-type [WT]) and -deficient (Il21r(-/-)) Ag-specific Tfh cells were used to help immunodeficient Il21r(-/-) B cells following T-dependent immunization. Il21r(-/-) B cells that had received help from WT Tfh cells, but not from Il21r(-/-) Tfh cells, generated affinity-matured Ab upon recall immunization. This effect was dependent on IL-4 produced in the primary response and associated with an increased fraction of memory B cells. Il21r(-/-) Tfh cells were distinguished from WT Tfh cells by a decreased frequency, reduced conjugate formation with B cells, increased expression of programmed cell death 1, and reduced production of IL-4. IL-21 also influenced responsiveness to IL-4 because expression of both membrane IL-4R and the IL-4-neutralizing soluble (s)IL-4R were reduced in Il21r(-/-) mice. Furthermore, the concentration of sIL-4R was found to correlate inversely with the amount of IgE in sera, such that the highest IgE levels were observed in Il21r(-/-) mice with the least sIL-4R. Taken together, these findings underscore the important collaboration between IL-4 and IL-21 in shaping T-dependent Ab responses.

Regulatory T cells prevent inducible BALT formation by dampening neutrophilic inflammation.

Inducible BALT (iBALT) can amplify pulmonary or systemic inflammatory responses to the benefit or detriment of the host. We took advantage of the age-dependent formation of iBALT to interrogate the underlying mechanisms that give rise to this ectopic, tertiary lymphoid organ. In this study, we show that the reduced propensity for weanling as compared with neonatal mice to form iBALT in response to acute LPS exposure is associated with greater regulatory T cell expansion in the mediastinal lymph nodes. Ab- or transgene-mediated depletion of regulatory T cells in weanling mice upregulated the expression of IL-17A and CXCL9 in the lungs, induced a tissue neutrophilia, and increased the frequency of iBALT to that observed in neonatal mice. Remarkably, neutrophil depletion in neonatal mice decreased the expression of the B cell active cytokines, a proliferation-inducing ligand and IL-21, and attenuated LPS-induced iBALT formation. Taken together, our data implicate a role for neutrophils in lymphoid neogenesis. Neutrophilic inflammation is a common feature of many autoimmune diseases in which iBALT are present and pathogenic, and hence the targeting of neutrophils or their byproducts may serve to ameliorate detrimental lymphoid neogenesis in a variety of disease contexts.

IL-21 contributes to fatal inflammatory disease in the absence of Foxp3+ T regulatory cells.

The cytokine IL-21 has been shown to influence immune responses through both costimulatory effects on effector T cells and opposing inhibitory effects on T regulatory cells (Tregs). To distinguish the effect of IL-21 on the immune system from that of its effect on Tregs, we analyzed the role of IL-21/IL-21R signaling in mice made genetically deficient in IL-2, which exhibit a deficit in IL-2-dependent Foxp3 regulatory T cells and suffer from a fatal multiorgan inflammatory disease. Our findings demonstrate that in the absence of IL-21/IL-21R signaling, Il2(-/-) mice retained a deficiency in Tregs yet exhibited a reduced and delayed inflammatory disease. The improved health of Il2(-/-)Il21r(-/-) mice was reflected in reduced pancreatitis and hemolytic anemia and this was associated with distinct changes in lymphocyte effector populations, including the reduced expansion of both T follicular helper cells and Th17 cells and a compensatory increase in IL-22 in the absence of IL-21R. IL-21/IL-21R interactions were also important for the expansion of effector and memory CD8(+) T cells, which were critical for the development of pancreatitis in Il2(-/-) mice. These findings demonstrate that IL-21 is a major target of immune system regulation.

Emerging cellular networks for regulation of T follicular helper cells.

The cellular networks that regulate humoral immune responses have been a focus of research over the past three decades. Studies have shown that inhibition of immune responses can be attributed to both suppressor T cells and B cells. More recently, T follicular helper (Tfh) cells have been identified as a target of immune regulation. Tfh cells are a subset of highly activated T helper cells specialized for providing cognate help to B cells during germinal center reactions. In this review, we describe emerging evidence for cellular networks that alter Tfh cell phenotype and function and regulate antibody production during the germinal center reaction. We discuss how these new findings influence our understanding of Tfh cells.

IL-21-producing Th cells in immunity and autoimmunity.

IL-21 is a member of the common γ-chain signaling family of cytokines. Analyses of the behavior of immune cells in response to IL-21 in vitro and studies of mice deficient in IL-21 or its receptor indicate that IL-21 has a role in lymphocyte activation, proliferation, differentiation, and survival. IL-21-producing CD4(+) Th cells constitute a broad array of helper subtypes including T follicular helper cells and Th17 cells. Both autocrine and paracrine utilization of IL-21 contributes to the overall signal transduction pathways of the Ag receptor to influence the growth and survival of lymphocytes. The redundancy that IL-21 exhibits in lymphoid organs during immune responses is in stark contrast to the evidence that pharmacological neutralization of this cytokine can halt inflammation in nonlymphoid organs where IL-21 becomes the dominant voice.

Calcineurin-dependent negative regulation of CD94/NKG2A expression on naive CD8+ T cells.

Immune responses lead to expression of immunoregulatory molecules on T cells, including natural killer (NK) receptors, such as CD94/NKG2A on CD8(+) T cells; these receptors restrain CD8(+) responses, thereby preventing T-cell exhaustion in chronic infections and limiting immunopathology. Here, we examined the requirements for inducing CD94/NKG2A on T cells responding to antigen. In vitro, moderate induction of CD94/NKG2A expression occurred after exposure of naive CD8(+) (but not CD4(+)) cells to CD3 ligation or specific peptide. Surprisingly, expression was inhibited by CD28/B7 costimulation. Such inhibition applied only to CD94/NKG2A and not other NK receptors (NKG2D) and was mediated by IL-2. Inhibition by IL-2 occurred via a NFAT cell-independent component of the calcineurin pathway, and CD94/NKG2A induction was markedly enhanced in the presence of calcineurin blockers, such as FK506 or using calcineurin-deficient T cells, both in vitro and in vivo. In addition to CD28-dependent inhibition by IL-2, CD94/NKG2A expression was impaired by several other cytokines (IL-4, IL-23, and transforming growth factor-ß) but enhanced by others (IL-6, IL-10, and IL-21). The complex interplay between these various stimuli may account for the variable expression of CD94/NKG2A during responses to different pathogens in vivo.

Protocol for the production and purification of an i-Motif-specific nanobody.

Intercalated motifs or i-Motifs (iMs) are nucleic acid structures formed by cytosine-rich sequences, which may regulate cellular processes and have broad applications in nanotechnology due to their pH-dependent nature. We have developed an iM-specific nanobody (iMbody) that can recognize iM DNA structures regardless of their sequences, making it a versatile research tool for studying iMs in various contexts. Here, we provide a protocol for the bacterial expression and His-tag purification of iMbody. We then describe procedures for performing ELISA and immunostaining using iMbody.

Loss of parity between IL-2 and IL-21 in the NOD Idd3 locus.

IL-2 and IL-21 are two cytokines with great potential to affect autoimmune infiltration of nonlymphoid tissue, and are contained within the strongest non-MHC-linked locus for type 1 diabetes (T1D) susceptibility on the nonobese diabetic (NOD) mouse (Idd3). IL-21 is necessary for the development of diabetes in the NOD mouse, but a number of important studies argue that decreased expression of IL-2 explains Idd3. In this study, we demonstrate that the amount of IL-21, but not IL-2, correlated with T1D incidence. During our analyses of the IL-2/IL-21 interval, we found that mice segregate into one of two distinct expression profiles. In the first group, which includes the C57BL/6 strain, both Il2 and Il21 were expressed at low levels. In the other group, which includes the NOD strain, Il2 and Il21 were both highly expressed. However, because NOD IL-2 mRNA was relatively unstable, IL-2 production was remarkably similar between strains. The increased production of IL-21 in NOD mice was found to result from two single nucleotide polymorphisms within the distal promoter region that conferred increased binding affinity for the transcription factor Sp1. Our findings indicate that a loss of locus parity after decreased IL-2 mRNA stability ensures that the high-expressing IL-21 allele persists in nature and provides a basis for autoimmunity.

RNA-seq and ChIP-seq Identification of Unique and Overlapping Targets of GLI Transcription Factors in Melanoma Cell Lines.

BACKGROUND: Despite significant progress in therapy, melanoma still has a rising incidence worldwide, and novel treatment strategies are needed. Recently, researchers have recognized the involvement of the Hedgehog-GLI (HH-GLI) signaling pathway in melanoma and its consistent crosstalk with the MAPK pathway. In order to further investigate the link between the two pathways and to find new target genes that could be considered for combination therapy, we set out to find transcriptional targets of all three GLI proteins in melanoma. METHODS: We performed RNA sequencing on three melanoma cell lines (CHL-1, A375, and MEL224) with overexpressed GLI1, GLI2, and GLI3 and combined them with the results of ChIP-sequencing on endogenous GLI1, GLI2, and GLI3 proteins. After combining these results, 21 targets were selected for validation by qPCR. RESULTS: RNA-seq revealed a total of 808 differentially expressed genes (DEGs) for GLI1, 941 DEGs for GLI2, and 58 DEGs for GLI3. ChIP-seq identified 527 genes that contained GLI1 binding sites in their promoters, 1103 for GLI2 and 553 for GLI3. A total of 15 of these targets were validated in the tested cell lines, 6 of which were detected by both RNA-seq and ChIP-seq. CONCLUSIONS: Our study provides insight into the unique and overlapping transcriptional output of the GLI proteins in melanoma. We suggest that our findings could provide new potential targets to consider while designing melanoma-targeted therapy.