John R. Ferdinand

Mechanisms of Jak/STAT Signaling in Immunity and Disease

More than two decades ago, experiments on the antiviral mechanisms of IFNs led to the discovery of JAKs and their downstream effectors, the STAT proteins. This pathway has since become a paradigm for membrane-to-nucleus signaling and explains how a broad range of soluble factors, including cytokines and hormones, mediate their diverse functions. Jak/STAT research has not only impacted basic science, particularly in the context of intercellular communication and cell-extrinsic control of gene expression, it also has become a prototype for transition from bench to bedside, culminating in the development and clinical implementation of pathway-specific therapeutics. This brief review synthesizes our current understanding of Jak/STAT biology while taking stock of the lessons learned and the challenges that lie ahead.

The TNF-Family Ligand TL1A and Its Receptor DR3 Promote T Cell–Mediated Allergic Immunopathology by Enhancing Differentiation and Pathogenicity of IL-9–Producing T Cells

The TNF family cytokine TL1A (Tnfsf15) costimulates T cells and type 2 innate lymphocytes (ILC2) through its receptor DR3 (Tnfrsf25). DR3-deficient mice have reduced T cell accumulation at the site of inflammation and reduced ILC2-dependent immune responses in a number of models of autoimmune and allergic diseases. In allergic lung disease models, immunopathology and local Th2 and ILC2 accumulation is reduced in DR3-deficient mice despite normal systemic priming of Th2 responses and generation of T cells secreting IL-13 and IL-4, prompting the question of whether TL1A promotes the development of other T cell subsets that secrete cytokines to drive allergic disease. In this study, we find that TL1A potently promotes generation of murine T cells producing IL-9 (Th9) by signaling through DR3 in a cell-intrinsic manner. TL1A enhances Th9 differentiation through an IL-2 and STAT5-dependent mechanism, unlike the TNF-family member OX40, which promotes Th9 through IL-4 and STAT6. Th9 differentiated in the presence of TL1A are more pathogenic, and endogenous TL1A signaling through DR3 on T cells is required for maximal pathology and IL-9 production in allergic lung inflammation. Taken together, these data identify TL1A–DR3 interactions as a novel pathway that promotes Th9 differentiation and pathogenicity. TL1A may be a potential therapeutic target in diseases dependent on IL-9.

The death receptor 3/TL1A pathway is essential for efficient development of antiviral CD4+ and CD8+ T-cell immunity

Death receptor 3 (DR3, TNFRSF25), the closest family relative to tumor necrosis factor receptor 1, promotes CD4+ T‐cell‐driven inflammatory disease. We investigated the in vivo role of DR3 and its ligand TL1A in viral infection, by challenging DR3‐deficient (DR3KO) mice and their DR3WT littermates with the β‐herpesvirus murine cytomegalovirus or the poxvirus vaccinia virus. The phenotype and function of splenic T‐cells were analyzed using flow cytometry and molecular biological techniques. We report surface expression of DR3 by naive CD8+ T cells, with TCR activation increasing its levels 4‐fold and altering the ratio of DR3 splice variants. T‐cell responses were reduced up to 90% in DR3KO mice during acute infection. Adoptive transfer experiments indicated this was dependent on T‐cell‐restricted expression of DR3. DR3‐dependent CD8+ T‐cell expansion was NK and CD4 independent and due to proliferation, not decreased cell death. Notably, impaired immunity in DR3KO hosts on a C57BL/6 background was associated with 4‐ to 7‐fold increases in viral loads during the acute phase of infection, and in mice with suboptimal NK responses was essential for survival (37.5%). This is the first description of DR3 regulating virus‐specific T‐cell function in vivo and uncovers a critical role for DR3 in mediating antiviral immunity.—Twohig, J. P., Marsden, M., Cuff, S. M., Ferdinand, J. R., Gallimore, A M., Perks, W. V., Al‐Shamkhani, A., Humphreys, I. R., Wang, E. C. Y. The death receptor 3/TL1A pathway is essential for efficient development of antiviral CD4+ and CD8+ T‐cell immunity. FASEB J. 26, 3575–3586 (2012). www.fasebj.org

Triggering of TNFRSF25 promotes CD8+ T‐cell responses and anti‐tumor immunity

TNFRSF25 is a member of the TNF receptor superfamily (TNFRSF) that binds to the TNF‐like protein TL1A. Although recent studies have demonstrated a role for TNFRSF25 in regulating CD4+ T‐cell responses, it remains to be determined if TNFRSF25 functions as a costimulatory receptor for CD8+ T cells. Here, we demonstrate that ectopic expression of TL1A on mouse plasmacytomas promotes elimination of tumor cells in a CD8+ T‐cell‐dependent manner and renders mice immune to a subsequent challenge with tumor cells. To gain further insight into the role of TNFRSF25 in CD8+ T‐cell responses, we analyzed the effect of TNFRSF25 triggering on OT‐I TCR transgenic T cells. We demonstrate that TNFRSF25 triggering in vivo with soluble TL1A promotes the proliferation and accumulation of antigen‐specific CD8+ T cells as well as their differentiation into CTLs. Furthermore, we show that TNFRSF25 also functions as a costimulatory receptor for memory CD8+ T cells. Thus, TNFRSF25 triggering enhances the secondary expansion of endogenous antigen‐specific memory CD8+ T cells. Our data suggest that TNFRSF25 agonists, such as soluble TL1A, could potentially be used to enhance the immunogenicity of vaccines that aim to elicit human anti‐tumor CD8+ T cells.

The TNF‐family cytokine TL1A: from lymphocyte costimulator to disease co‐conspirator

Originally described in 2002 as a T cell‐costimulatory cytokine, the tumor necrosis factor family member TNF‐like factor 1A (TL1A), encoded by the TNFSF15 gene, has since been found to affect multiple cell lineages through its receptor, death receptor 3 (DR3, encoded by TNFRSF25) with distinct cell‐type effects. Genetic deficiency or blockade of TL1A‐DR3 has defined a number of disease states that depend on this cytokine‐receptor pair, whereas excess TL1A leads to allergic gastrointestinal inflammation through stimulation of group 2 innate lymphoid cells. Noncoding variants in the TL1A locus are associated with susceptibility to inflammatory bowel disease and leprosy, predicting that the level of TL1A expression may influence host defense and the development of autoimmune and inflammatory diseases.

Expression of TNFRSF25 on conventional T cells and Tregs

Immunological tolerance is achieved through recessive and dominant mechanisms. In recessive tolerance the fate of self-reactive T cells is controlled in a cell intrinsic manner such that they undergo cell death or become anergic after exposure to self-antigen. In contrast, dominant tolerance is cell extrinsic and is mediated by CD4+ Tregs that express the transcription factor Forkhead P3 (Foxp3) and show increased self-reactivity when compared with conventional T cells (1). Peripheral homeostasis of Tregs is maintained through T cell receptor stimulation and signaling by IL-2 and costimulatory receptors such as CD28 (2, 3). Identifying the full spectrum of signals controlling Treg homeostasis in vivo may lend itself to therapeutic strategies that can be used to manipulate their number or function in order to ameliorate inflammatory diseases or augment an antitumor immune response. In this regard, the recent study by Schreiber and colleagues in the JCI (4) is particularly interesting, since the authors demonstrate that injection of mice with a mAb that binds to TNF receptor superfamily member 25 (TNFRSF25) selectively expands Tregs. The authors also show that administration of the anti-TNFRSF25 mAb inhibits a Th2 inflammatory response in the lungs of antigen-sensitized mice. Another interesting observation reported in that study was the preferential binding of the anti-TNFRSF25 mAb to Tregs (4). Based on this, the authors concluded that the level of TNFRSF25 on Tregs is considerably higher than that found on conventional CD4+ T cells. We have reexamined the expression of TNFRSF25 on conventional CD4+ T cells and Tregs, and our findings are remarkably different to those reported by Schreiber et al. We used two approaches to investigate expression of TNFRSF25 on T cell subsets (Figure ​(Figure1).1). First, we showed that a different anti-TNFRSF25 antibody stains conventional T cells and Tregs obtained from Foxp3-GFP knockin mice (5) with similar intensity (Figure ​(Figure1,1, A–C). Second, we demonstrated that soluble recombinant TL1A (sTL1A), the ligand for TNFRSF25, binds equally well to conventional CD4+ T cells and Tregs (Figure ​(Figure1D).1D). We therefore conclude that Tregs and conventional CD4+ T cells express similar levels of functional TNFRSF25. Figure 1 CD4+ conventional T cells and Tregs express similar levels of TNFRSF25. We agree with the findings of Schreiber et al. that TNFRSF25 triggering can expand Tregs. In fact, we recently showed that transgenic mice that constitutively express TL1A have increased numbers of Tregs (6). However, we also observed increased activation of conventional CD4+ T cells, elevated levels of IL-13 and IL-17, and small intestinal immune pathology that manifests as goblet cell and paneth cell hyperplasia (6). A similar phenotype was reported by Siegel and colleagues using mice that express higher levels of a TL1A transgene (7). Interestingly, we found that stimulation of T cells with recombinant TL1A attenuates Treg-mediated suppression in vitro and this effect required TNFRSF25 signaling in either conventional CD4+ T cells or Tregs (6). Thus, TNFRSF25 exerts costimulatory effects on conventional CD4+ T cells as well as on Tregs. Furthermore, its effects on promoting Treg turnover are counterbalanced by its ability to attenuate immune suppression and stimulate effector T cells. This conclusion is in line with findings obtained using mice that were rendered deficient in either TNFRSF25 or TL1A. In these mice, the net effect of defective TNFRSF25 signaling is a reduction in the severity of T cell–mediated inflammation (8, 9). How then can these findings been reconciled? We suspect that the anti-TNFRSF25 mAb used by Schreiber et al. may bind to an epitope that is preferentially expressed on Tregs, since multiple mRNA isoforms of TNFRSF25 have been described (10), and this may have lead to preferential expansion of Tregs in their experiments. Indeed, this notion is supported by a previous study showing differences in TNFRSF25 mRNA isoform expression between Tregs and Th17 cells (8). Thus, when compared with conventional T cells, Tregs expressed more TNFRSF25 mRNA transcripts that encode the shorter variant of TNFRSF25, which lacks the fourth extracellular cysteine-rich repeat. It is noteworthy that the antibody used in our study bound to both full-length TNFRSF25 as well as the short variant lacking the fourth cysteine-rich repeat (Figure ​(Figure1E).1E). We believe that caution should be applied in interpreting the findings obtained with anti-TNFRSF25 antibodies, as these may differ in their capacity to stimulate Tregs and effector T cells.

Renal Sodium Gradient Orchestrates a Dynamic Antibacterial Defense Zone

Lower urinary tract infections are among the most common human bacterial infections, but extension to the kidneys is rare. This has been attributed to mechanical forces, such as urine flow, that prevent the ascent of bladder microbes. Here, we show that the regional hypersalinity, required for the kidneys urine-concentrating function, instructs epithelial cells to produce chemokines that localize monocyte-derived mononuclear phagocytes (MNPs) to the medulla. This hypersaline environment also increases the intrinsic bactericidal and neutrophil chemotactic activities of MNPs to generate a zone of defense. Because MNP positioning and function are dynamically regulated by the renal salt gradient, we find that patients with urinary concentrating defects are susceptible to kidney infection. Our work reveals a critical accessory role for the homeostatic function of a vital organ in optimizing tissue defense.

Single-cell transcriptomes from human kidneys reveal the cellular identity of renal tumors

Pediatric and adult kidney tumors differ Understanding tumor origins and the similarities and differences between organ-specific cancers is important for determining treatment options. Young et al. generated more than 72,000 single-cell transcriptomes from healthy and cancerous human kidneys. From these data, they determined that Wilms tumor, a pediatric kidney cancer, originates from aberrant fetal cells, whereas adult kidney cancers are likely derived from a specific subtype of proximal convoluted tubular cell. Science, this issue p. 594 Single-cell mRNAs of normal and cancerous kidney cells reveal the cellular identity of childhood and adult tumors. Messenger RNA encodes cellular function and phenotype. In the context of human cancer, it defines the identities of malignant cells and the diversity of tumor tissue. We studied 72,501 single-cell transcriptomes of human renal tumors and normal tissue from fetal, pediatric, and adult kidneys. We matched childhood Wilms tumor with specific fetal cell types, thus providing evidence for the hypothesis that Wilms tumor cells are aberrant fetal cells. In adult renal cell carcinoma, we identified a canonical cancer transcriptome that matched a little-known subtype of proximal convoluted tubular cell. Analyses of the tumor composition defined cancer-associated normal cells and delineated a complex vascular endothelial growth factor (VEGF) signaling circuit. Our findings reveal the precise cellular identities and compositions of human kidney tumors.

Cleavage of TL1A Differentially Regulates Its Effects on Innate and Adaptive Immune Cells

TNF superfamily cytokines play major roles in the regulation of adaptive and innate immunity. The TNF superfamily cytokine TL1A (TNFSF15), through its cognate receptor DR3 (TNFRSF25), promotes T cell immunity to pathogens and directly costimulates group 2 and 3 innate lymphoid cells. Polymorphisms in the TNFSF15 gene are associated with the risk for various human diseases, including inflammatory bowel disease. Like other cytokines in the TNF superfamily, TL1A is synthesized as a type II transmembrane protein and cleaved from the plasma membrane by metalloproteinases. Membrane cleavage has been shown to alter or abrogate certain activities of other TNF family cytokines; however, the functional capabilities of membrane-bound and soluble forms TL1A are not known. Constitutive expression of TL1A in transgenic mice results in expansion of activated T cells and promotes intestinal hyperplasia and inflammation through stimulation of group 2 innate lymphoid cells. Through the generation of membrane-restricted TL1A-transgenic mice, we demonstrate that membrane TL1A promotes expression of inflammatory cytokines in the lung, dependent upon DR3 expression on T cells. Soluble TL1A alone was unable to produce this phenotype but was still able to induce intestinal type 2 inflammation independently of T cells. These data suggest differential roles for membrane and soluble TL1A on adaptive and innate immune cells and have implications for the consequences of blocking these two forms of TL1A.

Non-Invasive Multiphoton Imaging of Islets Transplanted Into the Pinna of the NOD Mouse Ear Reveals the Immediate Effect of Anti-CD3 Treatment in Autoimmune Diabetes.

We present a novel and readily accessible method facilitating cellular time-resolved imaging of transplanted pancreatic islets. Grafting of islets to the mouse ear pinna allows non-invasive, in vivo longitudinal imaging of events in the islets and enables improved acquisition of experimental data and use of fewer experimental animals than is possible using invasive techniques, as the same mouse can be assessed for the presence of islet infiltrating cells before and after immune intervention. We have applied this method to investigating therapeutic protection of beta cells through the well-established use of anti-CD3 injection, and have acquired unprecedented data on the nature and rapidity of the effect on the islet infiltrating T cells. We demonstrate that infusion of anti-CD3 antibody leads to immediate effects on islet infiltrating T cells in islet grafts in the pinna of the ear, and causes them to increase their speed and displacement within 20 min of infusion. This technique overcomes several technical challenges associated with intravital imaging of pancreatic immune responses and facilitates routine study of beta islet cell development, differentiation, and function in health and disease.