Structural insights into the assembly of gp130 family cytokine signaling complexes.

The interleukin-6 (IL-6) family cytokines signal through gp130 receptor homodimerization or heterodimerization with a second signaling receptor and play crucial roles in various cellular processes. We determined cryo-electron microscopy structures of five signaling complexes of this family, containing full receptor ectodomains bound to their respective ligands ciliary neurotrophic factor, cardiotrophin-like cytokine factor 1 (CLCF1), leukemia inhibitory factor, IL-27, and IL-6. Our structures collectively reveal similarities and differences in the assembly of these complexes. The acute bends at both signaling receptors in all complexes bring the membrane-proximal domains to a ~30 angstrom range but with distinct distances and orientations. We also reveal how CLCF1 engages its secretion chaperone cytokine receptor-like factor 1. Our data provide valuable insights for therapeutically targeting gp130-mediated signaling.

Comparison of the Inflammatory Circuits in Psoriasis Vulgaris, Non‒Pustular Palmoplantar Psoriasis, and Palmoplantar Pustular Psoriasis.

Palmoplantar pustular psoriasis (PPPP) and non‒pustular palmoplantar psoriasis (NPPP) are localized, debilitating forms of psoriasis. The inflammatory circuits involved in PPPP and NPPP are not well-understood. To compare the cellular and immunological features that differentiate PPPP and NPPP, skin biopsies were collected from a total of 30 participants with PPPP, NPPP, and psoriasis vulgaris (PV) and from 10 healthy participants. A subset consented to a second biopsy after 3 additional weeks off medication. Histologic staining of lesional and nonlesional skin showed higher neutrophil counts in PPPP than in NPPP and PV and higher CD8+ T-cell counts in NPPP. RNA sequencing and transcriptional analysis of skin biopsies showed enhanced IFN-γ pathway activation in NPPP lesions but stronger signatures of IL-17 pathway and neutrophil-related genes (e.g., IL36A) in PPPP lesional skin. Serum analysis on the Olink platform detected higher concentrations of T helper type 1, IFN-γ‒inducible chemokines in NPPP, and higher neutrophil-associated cytokines in PPPP. Taken together, this evidence suggests more pronounced T helper 1‒mediated inflammation in NPPP than in PV and PPPP and stronger neutrophil-associated activity in PPPP than in NPPP and PV. These data support targeting inflammatory pathways associated with neutrophilic inflammation (e.g., IL-36 signaling) for therapeutic development in PPPP.

Cancer cell-derived type I interferons instruct tumor monocyte polarization.

Monocytes are highly plastic immune cells that modulate antitumor immunity. Therefore, identifying factors that regulate tumor monocyte functions is critical for developing effective immunotherapies. Here, we determine that endogenous cancer cell-derived type I interferons (IFNs) control monocyte functional polarization. Guided by single-cell transcriptomic profiling of human and mouse tumors, we devise a strategy to distinguish and separate immunostimulatory from immunosuppressive tumor monocytes by surface CD88 and Sca-1 expression. Leveraging this approach, we show that cGAS-STING-regulated cancer cell-derived IFNs polarize immunostimulatory monocytes associated with anti-PD-1 immunotherapy response in mice. We also demonstrate that immunosuppressive monocytes convert into immunostimulatory monocytes upon cancer cell-intrinsic cGAS-STING activation. Consistently, we find that human cancer cells can produce type I IFNs that polarize monocytes, and our immunostimulatory monocyte gene signature is enriched in patient tumors that respond to anti-PD-1 immunotherapy. Our work exposes a role for cancer cell-derived IFNs in licensing monocyte functions that influence immunotherapy outcomes.

Development of Novel Glucocorticoids for Use in Antibody-Drug Conjugates for the Treatment of Inflammatory Diseases.

Glucocorticoids (GCs) are widely used to treat a variety of autoimmune and inflammatory diseases; however, systemic delivery of GCs is associated with side effects that affect essentially every organ system, reflecting the nearly ubiquitous expression of the glucocorticoid receptor (GR). Targeted delivery of GCs to diseased tissues using antibody-glucocorticoid conjugates (GC-ADCs) offers a therapeutic alternative to overcome these adverse effects. Herein, we describe novel classes of GCs that exhibited greater potency than dexamethasone and budesonide, a 100-fold selectivity toward the GR over other nuclear receptors, and no in vitro safety liability in pharmacology assays (hERG, AMES) and that demonstrated a substantial reduction in tumor necrosis factor-α (TNF-α) release in mice challenged with lipopolysaccharide (LPS). The site-specific conjugated GC-ADCs via cathepsin-cleavable linkers were highly stable in plasma and specifically released GCs in antigen-positive cells, suggesting that these novel GCs can serve as ADC payloads to treat autoimmune and inflammatory diseases.

Butyrophilin-like 2 regulates site-specific adaptations of intestinal γδ intraepithelial lymphocytes.

Tissue-resident γδ intraepithelial lymphocytes (IELs) orchestrate innate and adaptive immune responses to maintain intestinal epithelial barrier integrity. Epithelia-specific butyrophilin-like (Btnl) molecules induce perinatal development of distinct Vγ TCR+ IELs, however, the mechanisms that control γδ IEL maintenance within discrete intestinal segments are unclear. Here, we show that Btnl2 suppressed homeostatic proliferation of γδ IELs preferentially in the ileum. High throughput transcriptomic characterization of site-specific Btnl2-KO γδ IELs reveals that Btnl2 regulated the antimicrobial response module of ileal γδ IELs. Btnl2 deficiency shapes the TCR specificities and TCRγ/δ repertoire diversity of ileal γδ IELs. During DSS-induced colitis, Btnl2-KO mice exhibit increased inflammation and delayed mucosal repair in the colon. Collectively, these data suggest that Btnl2 fine-tunes γδ IEL frequencies and TCR specificities in response to site-specific homeostatic and inflammatory cues. Hence, Btnl-mediated targeting of γδ IEL development and maintenance may help dissect their immunological functions in intestinal diseases with segment-specific manifestations.

Absence of central tolerance in Aire-deficient mice synergizes with immune-checkpoint inhibition to enhance antitumor responses.

The endogenous anti-tumor responses are limited in part by the absence of tumor-reactive T cells, an inevitable consequence of thymic central tolerance mechanisms ensuring prevention of autoimmunity. Here we show that tumor rejection induced by immune checkpoint blockade is significantly enhanced in Aire-deficient mice, the epitome of central tolerance breakdown. The observed synergy in tumor rejection extended to different tumor models, was accompanied by increased numbers of activated T cells expressing high levels of Gzma, Gzmb, Perforin, Cxcr3, and increased intratumoural levels of Cxcl9 and Cxcl10 compared to wild-type mice. Consistent with Aire's central role in T cell repertoire selection, single cell TCR sequencing unveiled expansion of several clones with high tumor reactivity. The data suggest that breakdown in central tolerance synergizes with immune checkpoint blockade in enhancing anti-tumor immunity and may serve as a model to unmask novel anti-tumor therapies including anti-tumor TCRs, normally purged during central tolerance.

Enhanced IL-36R signaling promotes barrier impairment and inflammation in skin and intestine.

Deficiency in interleukin-36R (IL-36R) antagonist caused by loss-of-function mutations in IL-36RN leads to DITRA (deficiency of IL-36 receptor antagonist), a rare inflammatory human disease that belongs to a subgroup of generalized pustular psoriasis (GPP). We report a functional genetic mouse model of DITRA with enhanced IL-36R signaling analogous to that observed in patients with DITRA, which provides new insight into our understanding of the IL-36 family of molecules in regulating barrier integrity across multiple tissues. Humanized DITRA-like mice displayed increased skin inflammation in a preclinical model of psoriasis, and in vivo blockade of IL-36R pathway using anti-human IL-36R antibody ameliorated imiquimod-induced skin pathology as both prophylactic and therapeutic treatments. Deeper characterization of the humanized DITRA-like mice revealed that deregulated IL-36R signaling promoted tissue pathology during intestinal injury and led to impairment in mucosal restoration in the repair phase of chronic dextran sulfate sodium (DSS)-induced colitis. Blockade of IL-36R pathway significantly ameliorated DSS-induced intestinal inflammation and rescued the inability of DITRA-like mice to recover from mucosal damage in vivo. Our results indicate a central role for IL-36 in regulating proinflammatory responses in the skin and epithelial barrier function in the intestine, suggesting a new therapeutic potential for targeting the IL-36R axis in psoriasis and at the later stages of intestinal pathology in inflammatory bowel disease.

No differences observed among multiple clinical S1P1 receptor agonists (functional antagonists) in S1P1 receptor down-regulation and degradation.

Sphingosine-1-phosphate (S1P) is a bioactive metabolite with pleiotropic effects on multiple cellular processes in health and disease. Responses elicited by S1P are a result of binding to five specific G-protein-coupled receptors. We have developed multiple assays to systematically study the downstream signaling of these receptors, including early events such as direct receptor activation (GTPγS) as well as more distal events such as S1P1 receptor degradation. Employing such assays, we have characterized and compared multiple S1P1 agonists that are in clinical development including FTY720, BAF312, CS-0777, and other molecules from the S1P1 patent literature. Our parallel assessment has allowed us to compare their potency against S1P1, their selectivity against the four other S1P receptors, as well as species cross-reactivity. We note that all of the compounds studied signal in an identical manner through S1P1, leading to receptor degradation.

Circulating monocytes are reduced by sphingosine-1-phosphate receptor modulators independently of S1P3.

Sphingosine-1-phosphate (S1P) receptors are critical for lymphocyte egress from secondary lymphoid organs, and S1P receptor modulators suppress lymphocyte circulation. However, the role of S1P receptors on monocytes is less clear. To elucidate this, we systematically evaluated monocytes in rats and mice, both in naive and inflammatory conditions, with S1P receptor modulators FTY720 and BAF312. We demonstrate that S1P receptor modulators reduce circulating monocytes in a similar time course as lymphocytes. Furthermore, total monocyte numbers were increased in the spleen and bone marrow, suggesting that S1P receptor modulation restricts egress from hematopoietic organs. Monocytes treated ex vivo with FTY720 had reduced CD40 expression and TNF-α production, suggesting a direct effect on monocyte activation. Similar reductions in protein expression and cytokine production were also found in vivo. Suppression of experimental autoimmune encephalomyelitis in mice and rats by FTY720 correlated with reduced numbers of lymphocytes and monocytes. These effects on monocytes were independent of S1P3, as treatment with BAF312, a S1P1,4,5 modulator, led to similar results. These data reveal a novel role for S1P receptors on monocytes and offer additional insights on the mechanism of action of S1P receptor modulators in disease.

A multiply redundant genetic switch 'locks in' the transcriptional signature of regulatory T cells.

The transcription factor Foxp3 participates dominantly in the specification and function of Foxp3(+)CD4(+) regulatory T cells (T(reg) cells) but is neither strictly necessary nor sufficient to determine the characteristic T(reg) cell signature. Here we used computational network inference and experimental testing to assess the contribution of other transcription factors to this. Enforced expression of Helios or Xbp1 elicited distinct signatures, but Eos, IRF4, Satb1, Lef1 and GATA-1 elicited exactly the same outcome, acting in synergy with Foxp3 to activate expression of most of the T(reg) cell signature, including key transcription factors, and enhancing occupancy by Foxp3 at its genomic targets. Conversely, the T(reg) cell signature was robust after inactivation of any single cofactor. A redundant genetic switch thus 'locked in' the T(reg) cell phenotype, a model that would account for several aspects of T(reg) cell physiology, differentiation and stability.

The AKT-mTOR axis regulates de novo differentiation of CD4+Foxp3+ cells.

CD4(+)Foxp3(+) regulatory T (T reg) cells play an essential role in maintaining immunological tolerance via their suppressive function on conventional CD4(+) T (Tconv) cells. Repertoire studies suggest that distinct T cell receptor signaling pathways lead to T reg differentiation, but the signals that regulate T reg specification are largely unknown. We identify AKT as a strong repressor of entry into the T reg phenotype in vitro and in vivo. A constitutively active allele of AKT substantially diminished TGF-beta-induced Foxp3 expression in a kinase-dependent manner and via a rapamycin-sensitive pathway, implicating the AKT-mammalian target of rapamycin axis. The observed impairment in Foxp3 induction was part of a broad dampening of the typical T reg transcriptional signature. Expression of active AKT at a stage before Foxp3 turn on during normal T reg differentiation in the thymus selectively impaired differentiation of CD4(+)Foxp3(+) cells without any alteration in the positive selection of Tconv. Activated AKT, in contrast, did not affect established Foxp3 expression in T reg cells. These results place AKT at a nexus of signaling pathways whose proper activation has a strong and broad impact on the onset of T reg specification.

Foxp3 transcription-factor-dependent and -independent regulation of the regulatory T cell transcriptional signature.

The CD4(+)CD25(+) lineage of regulatory T (Treg) cells plays a key role in controlling immune and autoimmune responses and is characterized by a unique transcriptional signature. The transcription factor Foxp3 had been thought to determine the Treg cell lineage, a hypothesis challenged by recent observations. We have performed a cross-sectional analysis of the Treg cell signature in Treg-like cells generated under a number of conditions, with or without Foxp3, to delineate the elements that can be ascribed to T cell activation, interleukin-2, transforming growth factor-beta (TGF-beta) signaling, or Foxp3 itself. These influences synergized to determine many of the signature's components. Much of the Treg cell signature was not ascribable to Foxp3 because it contained gene clusters that are coregulated with, but not transactivated by, Foxp3. Thus, a higher level of regulation upstream of Foxp3 determines the lineage, distinct from elements downstream of Foxp3 that are essential for its regulatory properties.

Synergistic B cell activation by CD40 and the B cell antigen receptor: role of B lymphocyte antigen receptor-mediated kinase activation and tumor necrosis factor receptor-associated factor regulation.

Optimal activation of B-lymphocytes depends both upon expression of various cell surface receptors and adequate integration of signaling pathways. This requires signals generated upon recognition of antigen by the B lymphocyte antigen receptor (BCR) as well as additional signals provided by cognate interaction with T helper cells, including the CD40-CD154 interaction. Engagement of both the BCR and CD40 results in synergistic activation of B cells. Previous studies identified tumor necrosis factor receptor-associated factor (TRAF)-2 and TRAF3 in the CD40-signaling pathway together with BCR-activated protein kinase D (PKD) as important cooperative factors in this synergy. To better understand the role of these factors in bridging the BCR and CD40 signaling pathways, BCR signal regulation of TRAF function was examined. Results show that phosphorylation of TRAF2 is increased upon BCR but not CD40 engagement and that of the potentially phosphorylated residues of TRAF2, tyrosine 484 is crucial for BCR-CD40 synergy. Additionally, wild type or constitutively active Bruton's tyrosine kinase (Btk) enhanced, whereas the xid mutant form of Btk prevented, BCR-CD40 synergy. These effects were dependent upon TRAF2 and PKD activity. These findings suggest a model in which Btk contributes to the enhancement of the CD40 response by TRAF2 in a PKD-dependent manner.

A novel interaction between protein kinase D and TNF receptor-associated factor molecules regulates B cell receptor-CD40 synergy.

Signaling by Ag to the B cell Ag receptor (BCR) is enhanced by several cooperating signals, including several provided by B-T cell interactions. One of these, CD40, provides critical signals for B cell differentiation, isotype switching, and B cell memory. The molecular mechanisms by which BCR and CD40 signals synergize are not well understood. Although the BCR and CD40 share certain signaling pathways, we hypothesized that unique signals provided by each could provide mutual enhancement of their signaling pathways. The BCR, but not CD40, activates protein kinase D (PKD), while CD40, but not the BCR, employs the TNFR-associated factor (TRAF) adapter proteins in signaling. In this study, we show that genetic or pharmacologic inhibition of BCR-mediated PKD activation in B lymphocytes abrogated the synergy between the CD40 and the BCR, as measured by activation of Ig and cytokine secretion. Interestingly, the role of PKD was dependent upon the association of CD40 with TRAF2, and was inhibited by the binding of TRAF3, revealing a novel functional link between these two classes of signaling molecules.

Antigen-specific B-lymphocyte activation.

B lymphocytes comprise a major component of the adaptive mammalian immune system, having the exclusive ability to produce and secrete immunoglobulins (Ig) of various forms (isotypes). This property alone renders B-cell activation critical to immunity, but the fully activated B cell also functions in antigen presentation and the production of a variety of cytokines and chemokines. There are many signals that must be coordinated to achieve and properly regulate antigen-specific B-lymphocyte activation and the development of humoral memory. This review seeks to summarize these components, and to highlight recent advances in each area that contribute to a greater understanding of the complex processes involved in B-cell activation.

Tumor necrosis factor receptor-associated factor 2 (TRAF2)-deficient B lymphocytes reveal novel roles for TRAF2 in CD40 signaling.

CD40 function is initiated by tumor necrosis factor (TNF) receptor-associated factor (TRAF) adapter proteins, which play important roles in signaling by numerous receptors. Characterizing roles of individual TRAFs has been hampered by limitations of available experimental models and the poor viability of most TRAF-deficient mice. Here, B cell lines made deficient in TRAF2 using a novel homologous recombination system reveal new roles for TRAF2. We demonstrate that TRAF2 participates in synergy between CD40 and B cell antigen receptor signals, and in CD40-mediated, TNF-dependent IgM production. We also find that TRAF2 participates in the degradation of TRAF3 associated with CD40 signaling, a role that may limit inhibitory actions of TRAF3. Finally, we show that TRAF2 and TRAF6 have overlapping functions in CD40-mediated NF-kappaB activation and CD80 up-regulation. These findings demonstrate previously unappreciated roles for TRAF2 in signaling by TNF receptor family members, using an approach that facilitates the analysis of genes critical to the viability of whole organisms.

Cutting edge: molecular mechanisms of synergy between CD40 and the B cell antigen receptor: role for TNF receptor-associated factor 2 in receptor interaction.

Optimal Ag-specific B lymphocyte activation requires both recognition of Ag by the B cell Ag receptor (BCR) and contact-mediated interactions with Ag-specific Th lymphocytes. One of these interactions involves ligation of B cell CD40 by T cell-expressed CD154. CD40 signaling is crucial for Ab production, isotype switching, up-regulation of surface molecules, development of germinal centers, and the humoral memory response. The signaling pathways emanating from the BCR and CD40 are able to cooperate, but the molecular mechanisms responsible for this interaction are incompletely understood. The present study explored the roles of signaling motifs in the CD40 cytoplasmic tail in this synergy. We find that threonine in the PXQXT motif in the TNFR-associated factor-2 binding site is critical for synergistic effects of CD40 and BCR signals, independent of its phosphorylation. Furthermore, data suggest an indirect role for TNFR-associated factor-2 in the cooperative signaling.

Inhibitory oligonucleotides specifically block effects of stimulatory CpG oligonucleotides in B cells.

Reaction to certain motifs in bacterial DNA is an important function of natural immunity. For example, single stranded oligonucleotides (ODN) containing the motif "not C, unmethylated C, G, not G" are powerful mitogens and apoptosis inhibitors for mouse spleen B cells. But replacing GCGTT or ACGTT with GCGGG or ACGGG converted a stimulatory 15-mer ODN into an inhibitory ODN. All inhibitory ODN had three consecutive G, and a fourth G increased inhibitory activity, but a deazaguanosine substitution to prevent planar stacking did not affect activity. Inhibitory ODN blocked apoptosis protection and cell-cycle entry induced by stimulatory ODN, but not that induced by lipopolysaccharide, anti-CD40 or anti-IgM+IL-4. ODN-driven up-regulation of cyclin D(2), c-Myc, c-Fos, c-Jun and Bcl(XL) and down-regulation of cyclin kinase inhibitor p27(kip1) were all blocked by inhibitory ODN. The relative potency of a series of stimulatory and inhibitory ODN was the same for all readouts measured. Interference with uptake of stimulatory ODN could not account for their inhibitory effects. Even if addition of inhibitory ODN was delayed several hours, partial inhibition of stimulatory ODN effects occurred. Inhibitory ODN hold potential as antidotes for excessive ODN stimulation in the clinical setting and provide an important tool for studying ODN recognition.