Recruitment of calcineurin to the TCR positively regulates T cell activation.

Calcineurin is a phosphatase whose primary targets in T cells are NFAT transcription factors, and inhibition of calcineurin activity by treatment with cyclosporin A (CsA) or FK506 is a cornerstone of immunosuppressive therapies. Here we found that calcineurin was recruited to the T cell antigen receptor (TCR) signaling complex, where it reversed inhibitory phosphorylation of the tyrosine kinase Lck on Ser59 (LckS59). Loss of calcineurin activity impaired phosphorylation of Tyr493 of the tyrosine kinase ZAP-70 (ZAP-70Y493), as well as some downstream pathways in a manner consistent with signaling in cells expressing LckS59A (Lck that cannot be phosphorylated) or LckS59E (a phosphomimetic mutant). Notably, CsA inhibited integrin-LFA-1-dependent and NFAT-independent adhesion of T cells to the intercellular adhesion molecule ICAM-1, with little effect on cells expressing mutant Lck. These results provide new understanding of how widely used immunosuppressive drugs interfere with essential processes in the immune response.

The histone methyltransferase KMT2D maintains cellular glucocorticoid responsiveness by shielding the glucocorticoid receptor from degradation.

Because of their ability to induce lymphocyte apoptosis, glucocorticoids (GC) are widely used to treat hematological malignancies such as lymphomas and multiple myeloma. Their effectiveness is often limited, however, due to the development of glucocorticoid resistance by a variety of molecular mechanisms. Here we performed an unbiased genome-wide CRISPR screen with the human T-cell leukemia cell line Jurkat to find previously unidentified genes required for GC-induced apoptosis. One such gene was KMT2D (also known as MLL2 or MLL4), which encodes a histone lysine methyltransferase whose mutations are associated with a variety of cancers, blood malignancies in particular, and are considered markers of poor prognosis. Knockout of KMT2D by CRISPR/Cas9 gene editing in Jurkat and several multiple myeloma cell lines downregulated GR protein expression. Surprisingly, this was not due to a reduction in GR transcripts, but rather to a decrease in the protein's half-life, primarily due to proteasomal degradation. Reconstitution of KMT2D expression restored GR levels. In contrast to the known ability of KMT2D to control gene transcription through covalent histone methylation, KMT2D-mediated upregulation of GR levels did not require its methyltransferase activity. Co-immunoprecipitation and proximity ligation assays found constitutive binding of KMT2D to the GR, which was enhanced in the presence of GC. These observations reveal KMT2D to be essential for the stabilization of cellular GR levels, and suggest a possible mechanism by which KMT2D mutations may lead to GC resistance in some malignancies.

TNF plays a crucial role in inflammation by signaling via T cell TNFR2.

TNF, produced largely by T and innate immune cells, is potently proinflammatory, as are cytokines such as IFN-γ and IL-17 produced by Th1 and Th17 cells, respectively. Here, we asked if TNF is upstream of Th skewing toward inflammatory phenotypes. Exposure of mouse CD4+ T cells to TNF and TGF-ß generated Th17 cells that express low levels of IL-17 (ROR-γt+IL-17lo) and high levels of inflammatory markers independently of IL-6 and STAT3. This was mediated by the nondeath TNF receptor TNFR2, which also contributed to the generation of inflammatory Th1 cells. Single-cell RNA sequencing of central nervous system-infiltrating CD4+ T cells in mouse experimental autoimmune encephalomyelitis (EAE) found an inflammatory gene expression profile similar to cerebrospinal fluid-infiltrating CD4+ T cells from patients with multiple sclerosis. Notably, TNFR2-deficient CD4+ T cells produced fewer inflammatory mediators and were less pathogenic in EAE and colitis. IL-1ß, a Th17-skewing cytokine, induced TNF and proinflammatory granulocyte-macrophage colony-stimulating factor (GM-CSF) in T cells, which was inhibited by disruption of TNFR2 signaling, demonstrating IL-1ß can function indirectly via the production of TNF. Thus, TNF is not just an effector but also an initiator of inflammatory Th differentiation.

Unique properties of TCR-activated p38 are necessary for NFAT-dependent T-cell activation.

Nuclear factor of activated T cells (NFAT) transcription factors are required for induction of T-cell cytokine production and effector function. Although it is known that activation via the T-cell antigen receptor (TCR) results in 2 critical steps, calcineurin-mediated NFAT1 dephosphorylation and NFAT2 up-regulation, the molecular mechanisms underlying each are poorly understood. Here we find that T cell p38, which is activated by an alternative pathway independent of the mitogen-activated protein (MAP) kinase cascade and with different substrate specificities, directly controls these events. First, alternatively (but not classically) activated p38 was required to induce the expression of the AP-1 component c-Fos, which was necessary for NFAT2 expression and cytokine production. Second, alternatively (but not classically) activated p38 phosphorylated NFAT1 on a heretofore unidentified site, S79, and in its absence NFAT1 was unable to interact with calcineurin or migrate to the nucleus. These results demonstrate that the acquisition of unique specificities by TCR-activated p38 orchestrates NFAT-dependent T-cell functions.

Glucocorticoids Oppose Thymocyte Negative Selection by Inhibiting Helios and Nur77.

Glucocorticoid (GC) signaling in thymocytes shapes the TCR repertoire by antagonizing thymocyte negative selection. The transcription factors Nur77 and Helios, which are upregulated in TCR-signaled thymocytes, have been implicated in negative selection. In this study, we found that GCs inhibited Helios and, to a lesser extent, Nur77 upregulation in TCR-stimulated mouse thymocytes. Inhibition was increased by GC preincubation, and reductions in mRNA were prevented by a protein synthesis inhibitor, suggesting that GCs suppress indirectly via an intermediary factor. Upregulation of Helios in TCR-stimulated thymocytes was unaffected by deletion of Nur77, indicating Nur77 and Helios are regulated independently. Whereas CD4+ thymocytes are positively selected in wild-type AND TCR-transgenic B6 mice, loss of GC receptor expression resulted in increased negative selection. Correspondingly, Helios and Nur77 levels were elevated in TCRhiCD4+CD8+ (TCR-signaled) thymocytes. Notably, deletion of Helios fully reversed this negative selection, whereas deletion of Nur77 had no effect on CD4+CD8+ cell numbers but reversed the loss of mature CD4+ thymocytes. Thus, Nur77 and Helios are GC targets that play nonredundant roles in setting the signaling threshold for thymocyte negative selection.

Intensity and duration of TCR signaling is limited by p38 phosphorylation of ZAP-70T293 and destabilization of the signalosome.

ZAP-70 is a tyrosine kinase that is essential for initiation of T cell antigen receptor (TCR) signaling. We have found that T cell p38 MAP kinase (MAPK), which is directly phosphorylated and activated by ZAP-70 downstream of the TCR, in turn phosphorylates Thr-293 in the interdomain B region of ZAP-70. Mutant T cells expressing ZAP-70 with an alanine substitution at this residue (ZAP-70T293A) had enhanced TCR proximal signaling and increased effector responses. Lack of ZAP-70T293 phosphorylation increased association of ZAP-70 with the TCR and prolonged the existence of TCR signaling microclusters. These results identify a tight negative feedback loop in which ZAP-70-activated p38 reciprocally phosphorylates ZAP-70 and destabilizes the signaling complex.

Cutting Edge: De Novo Glucocorticoid Synthesis by Thymic Epithelial Cells Regulates Antigen-Specific Thymocyte Selection.

Glucocorticoid (GC) signaling in thymocytes counters negative selection and promotes the generation of a self-tolerant yet Ag-responsive T cell repertoire. Whereas circulating GC are derived from the adrenals, GC are also synthesized de novo in the thymus. The significance of this local production is unknown. In this study we deleted 11ß-hydroxylase, the enzyme that catalyzes the last step of GC biosynthesis, in thymic epithelial cells (TEC) or thymocytes. Like GC receptor-deficient T cells, T cells from mice lacking TEC-derived but not thymocyte-derived GC proliferated poorly to alloantigen, had a reduced antiviral response, and exhibited enhanced negative selection. Strikingly, basal expression of GC-responsive genes in thymocytes from mice lacking TEC-derived GC was reduced to the same degree as in GC receptor-deficient thymocytes, indicating that at steady-state the majority of biologically active GC are paracrine in origin. These findings demonstrate the importance of extra-adrenal GC even in the presence of circulating adrenal-derived GC.

Correction: Non-Canonical NF-κB Activation and Abnormal B Cell Accumulation in Mice Expressing Ubiquitin Protein Ligase-Inactive c-IAP2.

[This corrects the article DOI: 10.1371/journal.pbio.1000518.].

Host-Derived CD70 Suppresses Murine Graft-versus-Host Disease by Limiting Donor T Cell Expansion and Effector Function.

Allogeneic hematopoietic cell transplantation (allo-HCT) is a potentially curative treatment for hematologic and immunologic diseases. However, graft-versus-host disease (GVHD) may develop when donor-derived T cells recognize and damage genetically distinct normal host tissues. In addition to TCR signaling, costimulatory pathways are involved in T cell activation. CD27 is a TNFR family member expressed on T cells, and its ligand, CD70, is expressed on APCs. The CD27/CD70 costimulatory pathway was shown to be critical for T cell function and survival in viral infection models. However, the role of this pathway in allo-HCT is previously unknown. In this study, we have examined its contribution in GVHD pathogenesis. Surprisingly, Ab blockade of CD70 after allo-HCT significantly increases GVHD. Interestingly, whereas donor T cell- or bone marrow-derived CD70 plays no role in GVHD, host-derived CD70 inhibits GVHD as CD70-/- hosts show significantly increased GVHD. This is evidenced by reduced survival, more severe weight loss, and increased histopathologic damage compared with wild-type hosts. In addition, CD70-/- hosts have higher levels of proinflammatory cytokines TNF-α, IFN-γ, IL-2, and IL-17. Moreover, accumulation of donor CD4+ and CD8+ effector T cells is increased in CD70-/- versus wild-type hosts. Mechanistic analyses suggest that CD70 expressed by host hematopoietic cells is involved in the control of alloreactive T cell apoptosis and expansion. Together, our findings demonstrate that host CD70 serves as a unique negative regulator of allogeneic T cell response by contributing to donor T cell apoptosis and inhibiting expansion of donor effector T cells.

A20: more than one way to skin a cat.

In this issue of Molecular Cell, Skaug et al. (2011) propose a polyubiquitin-dependent, noncatalytic mechanism by which the deubiquitinase A20 inhibits IκB kinase and NF-κB activation.

Recruitment of A20 by the C-terminal domain of NEMO suppresses NF-κB activation and autoinflammatory disease.

Receptor-induced NF-κB activation is controlled by NEMO, the NF-κB essential modulator. Hypomorphic NEMO mutations result in X-linked ectodermal dysplasia with anhidrosis and immunodeficiency, also referred to as NEMO syndrome. Here we describe a distinct group of patients with NEMO C-terminal deletion (ΔCT-NEMO) mutations. Individuals harboring these mutations develop inflammatory skin and intestinal disease in addition to ectodermal dysplasia with anhidrosis and immunodeficiency. Both primary cells from these patients, as well as reconstituted cell lines with this deletion, exhibited increased IκB kinase (IKK) activity and production of proinflammatory cytokines. Unlike previously described loss-of-function mutations, ΔCT-NEMO mutants promoted increased NF-κB activation in response to TNF and Toll-like receptor stimulation. Investigation of the underlying mechanisms revealed impaired interactions with A20, a negative regulator of NF-κB activation, leading to prolonged accumulation of K63-ubiquitinated RIP within the TNFR1 signaling complex. Recruitment of A20 to the C-terminal domain of NEMO represents a novel mechanism limiting NF-κB activation by NEMO, and its absence results in autoinflammatory disease.

Suppression of Dendritic Cell-Derived IL-12 by Endogenous Glucocorticoids Is Protective in LPS-Induced Sepsis.

Sepsis, an exaggerated systemic inflammatory response, remains a major medical challenge. Both hyperinflammation and immunosuppression are implicated as causes of morbidity and mortality. Dendritic cell (DC) loss has been observed in septic patients and in experimental sepsis models, but the role of DCs in sepsis, and the mechanisms and significance of DC loss, are poorly understood. Here, we report that mice with selective deletion of the glucocorticoid receptor (GR) in DCs (GR(CD11c-cre)) were highly susceptible to LPS-induced septic shock, evidenced by elevated inflammatory cytokine production, hypothermia, and mortality. Neutralizing anti-IL-12 antibodies prevented hypothermia and death, demonstrating that endogenous GC-mediated suppression of IL-12 is protective. In LPS-challenged GR(CD11c-cre) mice, CD8(+) DCs were identified as the major source of prolonged IL-12 production, which correlated with elevations of NK cell-derived IFN-γ. In addition, the loss of GR in CD11c(+) cells rescued LPS-induced loss of CD8(+) DCs but not other DC subsets. Unlike wild-type animals, exposure of GR(CD11c-cre) mice to low-dose LPS did not induce CD8(+) DC loss or tolerance to subsequent challenge with high dose, but neutralization of IL-12 restored the ability of low-dose LPS to tolerize. Therefore, endogenous glucocorticoids blunt LPS-induced inflammation and promote tolerance by suppressing DC IL-12 production.

Live cell imaging unveils multiple domain requirements for in vivo dimerization of the glucocorticoid receptor.

Glucocorticoids are essential for life, but are also implicated in disease pathogenesis and may produce unwanted effects when given in high doses. Glucocorticoid receptor (GR) transcriptional activity and clinical outcome have been linked to its oligomerization state. Although a point mutation within the GR DNA-binding domain (GRdim mutant) has been reported as crucial for receptor dimerization and DNA binding, this assumption has recently been challenged. Here we have analyzed the GR oligomerization state in vivo using the number and brightness assay. Our results suggest a complete, reversible, and DNA-independent ligand-induced model for GR dimerization. We demonstrate that the GRdim forms dimers in vivo whereas adding another mutation in the ligand-binding domain (I634A) severely compromises homodimer formation. Contrary to dogma, no correlation between the GR monomeric/dimeric state and transcriptional activity was observed. Finally, the state of dimerization affected DNA binding only to a subset of GR binding sites. These results have major implications on future searches for therapeutic glucocorticoids with reduced side effects.

Structural basis for recognition of diubiquitins by NEMO.

NEMO is the regulatory subunit of the IkappaB kinase (IKK) in NF-kappaB activation, and its CC2-LZ region interacts with Lys63 (K63)-linked polyubiquitin to recruit IKK to receptor signaling complexes. In vitro, CC2-LZ also interacts with tandem diubiquitin. Here we report the crystal structure of CC2-LZ with two dimeric coiled coils representing CC2 and LZ, respectively. Surprisingly, mutagenesis and nuclear magnetic resonance experiments reveal that the binding sites for diubiquitins at LZ are composites of both chains and that each ubiquitin in diubiquitins interacts with symmetrical NEMO asymmetrically. For tandem diubiquitin, the first ubiquitin uses the conserved hydrophobic patch and the C-terminal tail, while the second ubiquitin uses an adjacent surface patch. For K63-linked diubiquitin, the proximal ubiquitin uses its conserved hydrophobic patch, while the distal ubiquitin mostly employs the C-terminal arm including the K63 linkage residue. These studies uncover the energetics and geometry for mutual recognition of NEMO and diubiquitins.

The many paths to p38 mitogen-activated protein kinase activation in the immune system.

Signals emanating from many cell-surface receptors and environmental cues converge on mitogen-activated protein kinases (MAPKs), which in turn phosphorylate and activate various transcription factors and other molecular effectors. Members of the p38 MAPK family, which respond to pro-inflammatory cytokines and cellular stresses, are typically activated by serial phosphorylation and activation of upstream kinases (the MAPK cascade). In this Review, I highlight the recent studies that indicate that p38-subfamily members can also be activated by non-canonical mechanisms, at least one of which seems to have an important role in antigen-receptor-activated T cells. These alternative pathways might have particular relevance for cells that participate in immune and inflammatory responses.

CYLD and the NEMO Zinc Finger Regulate Tumor Necrosis Factor Signaling and Early Embryogenesis.

NF-κB essential modulator (NEMO) and cylindromatosis protein (CYLD) are intracellular proteins that regulate the NF-κB signaling pathway. Although mice with either CYLD deficiency or an alteration in the zinc finger domain of NEMO (K392R) are born healthy, we found that the combination of these two gene defects in double mutant (DM) mice is early embryonic lethal but can be rescued by the absence of TNF receptor 1 (TNFR1). Notably, NEMO was not recruited into the TNFR1 complex of DM cells, and consequently NF-κB induction by TNF was severely impaired and DM cells were sensitized to TNF-induced cell death. Interestingly, the TNF signaling defects can be fully rescued by reconstitution of DM cells with CYLD lacking ubiquitin hydrolase activity but not with CYLD mutated in TNF receptor-associated factor 2 (TRAF2) or NEMO binding sites. Therefore, our data demonstrate an unexpected non-catalytic function for CYLD as an adapter protein between TRAF2 and the NEMO zinc finger that is important for TNF-induced NF-κB signaling during embryogenesis.

c-IAP ubiquitin protein ligase activity is required for 4-1BB signaling and CD8(+) memory T-cell survival.

Cellular inhibitor of apoptosis proteins (c-IAP) 1 and 2 are widely expressed ubiquitin protein ligases that regulate a variety of cellular functions, including the sensitivity of T cells to costimulation. 4-1BB is a TNF receptor family member that signals via a complex that includes TRAF family members and the c-IAPs to upregulate NF-κB and ERK, and has been implicated in memory T-cell survival. Here, we show that effector and memory T cells from mice expressing a dominant negative E3-inactive c-IAP2 (c-IAP2(H570A)) have impaired signaling downstream of 4-1BB. When infected with lymphocytic choriomeningitis virus, unlike mice in which c-IAPs were acutely downregulated by c-IAP antagonists, the primary response of c-IAP2(H570A) mice was normal. However, the number of antigen-specific CD8(+) but not CD4(+) T cells declined more rapidly and to a greater extent in c-IAP2(H570A) mice than in WT controls. Studies with T-cell adoptive transfer demonstrated that the enhanced decay of memory cells was T-cell intrinsic. Thus, c-IAP E3 activity is required for 4-1BB coreceptor signaling and maintenance of CD8(+) T-cell memory.

Dual function of CD70 in viral infection: modulator of early cytokine responses and activator of adaptive responses.

The role of the TNF family member CD70 in adaptive T cell responses has been intensively studied, but its function in innate responses is still under investigation. In this study, we show that CD70 inhibits the early innate response to murine CMV (MCMV) but is essential for the optimal generation of virus-specific CD8 T cells. CD70(-/-) mice reacted to MCMV infection with a robust type I IFN and proinflammatory cytokine response. This response was sufficient for initial control of MCMV, although at later time points, CD70(-/-) mice became more susceptible to MCMV infection. The heightened cytokine response during the early phase of MCMV infection in CD70(-/-) mice was paralleled by a reduction in regulatory T cells (Treg). Treg from naive CD70(-/-) mice were not as efficient at suppressing T cell proliferation compared with Treg from naive wild-type mice, and depletion of Treg during MCMV infection in Foxp3-diphtheria toxin receptor mice or in wild-type mice recapitulated the phenotype observed in CD70(-/-) mice. Our study demonstrates that although CD70 is required for the activation of the antiviral adaptive response, it has a regulatory role in early cytokine responses to viruses such as MCMV, possibly through maintenance of Treg survival and function.

IAPs: what's in a name?

Originally described in insect viruses, cellular proteins with Baculoviral IAP repeat (BIR) motifs have been thought to function primarily as inhibitors of apoptosis. The subsequent finding that a subset of IAPs that contain a RING domain have ubiquitin protein ligase (E3) activity implied the presence of other functions. It is now known that IAPs are involved in mitotic chromosome segregation, cellular morphogenesis, copper homeostasis, and intracellular signaling. Here, we review the current understanding of the roles of IAPs in apoptotic and nonapoptotic processes and explore the notion that the latter represents the primary physiologic activities of IAPs.