Centrosome amplification and aneuploidy driven by the HIV-1-induced Vpr•VprBP•Plk4 complex in CD4+ T cells.

HIV-1 infection elevates the risk of developing various cancers, including T-cell lymphoma. Whether HIV-1-encoded proteins directly contribute to oncogenesis remains unknown. We observe that approximately 1-5% of CD4+ T cells from the blood of people living with HIV-1 exhibit over-duplicated centrioles, suggesting that centrosome amplification underlies the development of HIV-1-associated cancers by driving aneuploidy. Through affinity purification, biochemical, and cellular analyses, we discover that Vpr, an accessory protein of HIV-1, hijacks the centriole duplication machinery and induces centrosome amplification and aneuploidy. Mechanistically, Vpr forms a cooperative ternary complex with an E3 ligase subunit, VprBP, and polo-like kinase 4 (Plk4). Unexpectedly, however, the complex enhances Plk4's functionality by promoting its relocalization to the procentriole assembly and induces centrosome amplification. Loss of either Vpr's C-terminal 17 residues or VprBP acidic region, the two elements required for binding to Plk4 cryptic polo-box, abrogates Vpr's capacity to induce these events. Furthermore, HIV-1 WT, but not its Vpr mutant, induces multiple centrosomes and aneuploidy in human primary CD4+ T cells. We propose that the Vpr•VprBP•Plk4 complex serves as a molecular link that connects HIV-1 infection to oncogenesis and that inhibiting the Vpr C-terminal motif may reduce the occurrence of HIV-1-associated cancers.

Centrosome amplification and aneuploidy driven by the HIV-1-induced Vpr•VprBP•Plk4 complex in CD4+ T cells.

HIV-1 infection elevates the risk of developing various cancers, including T-cell lymphoma. Whether HIV-1-encoded proteins directly contribute to oncogenesis remains unknown. We observed that approximately 1-5% of CD4+ T cells from the blood of people living with HIV-1 exhibit over-duplicated centrioles, suggesting that centrosome amplification underlies the development of HIV-1-associated cancers by driving aneuploidy. Through affinity purification, biochemical, and cell biology analyses, we discovered that Vpr, an accessory protein of HIV-1, hijacks the centriole duplication machinery and induces centrosome amplification and aneuploidy. Mechanistically, Vpr formed a cooperative ternary complex with an E3 ligase subunit, VprBP, and polo-like kinase 4 (Plk4). Unexpectedly, however, the complex enhanced Plk4's functionality by promoting its relocalization to the procentriole assembly and induced centrosome amplification. Loss of either Vpr's C-terminal 17 residues or VprBP acidic region, the two elements required for binding to Plk4 cryptic polo-box, abrogated Vpr's capacity to induce all these events. Furthermore, HIV-1 WT, but not its Vpr mutant, induced multiple centrosomes and aneuploidy in primary CD4+ T cells. We propose that the Vpr•VprBP•Plk4 complex serves as a molecular link that connects HIV-1 infection to oncogenesis and that inhibiting the Vpr C-terminal motif may reduce the occurrence of HIV-1-associated cancers.

Aire drives steroid hormone biosynthesis by medullary thymic epithelial cells.

Thymic epithelial cells (TECs) produce glucocorticoids, which antagonize negative selection of autoreactive thymocytes and promote a competent T cell antigen-specific repertoire. To characterize their source, we generated a knock-in reporter mouse in which endogenous Cyp11b1, the final enzyme in de novo production of active glucocorticoids, was fluorescently tagged with mScarlet. Here, we find that Cyp11b1 is expressed in medullary TECs (mTECs) but not cortical TECs or other cells in the thymus. A distinct characteristic of mTECs is the presence of Aire, a transcription factor that drives expression of tissue-restricted antigens (TRAs) important for establishing immune tolerance. Cyp11b1 expression was highest in Aire+ mTECs, lower in post-Aire mTECs, and absent in mTECs of Aire-deficient mice. Transcriptomic analyses found that multiple enzymatic biosynthetic pathways are expressed specifically in mTECs and are also Aire dependent. In particular, we found that the thymus expresses messenger RNA for enzymes that catalyze production of many bioactive steroids and that glucocorticoids and sex steroids were secreted by cultured thymi. Expression of the transcripts for these genes and production of their final steroid products were markedly reduced in the absence of Aire. Thus, in addition to its well-established role in inducing TRAs that promote negative selection, Aire has an additional and contrary function of inducing glucocorticoids that antagonize negative selection, which together may expand and enhance the TCR repertoire. Furthermore, because Aire drives expression of multiple enzymes responsible for production of other non-gene-encoded bioactive molecules, it might have yet other roles in thymus development and function.

Tumors produce glucocorticoids by metabolite recycling, not synthesis, and activate Tregs to promote growth.

Glucocorticoids are steroid hormones with potent immunosuppressive properties. Their primary source is the adrenals, where they are generated via de novo synthesis from cholesterol. In addition, many tissues have a recycling pathway in which glucocorticoids are regenerated from inactive metabolites by the enzyme 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1, encoded by Hsd11b1). Here, we find that multiple tumor types express Hsd11b1 and produce active glucocorticoids. Genetic ablation of Hsd11b1 in such cells had no effect on in vitro growth, but reduced in vivo tumor progression, which corresponded with increased frequencies of CD8+ tumor-infiltrating lymphocytes (TILs) expressing activation markers and producing effector cytokines. Tumor-derived glucocorticoids were found to promote signatures of Treg activation and suppress signatures of conventional T cell activation in tumor-infiltrating Tregs. Indeed, CD8+ T cell activation was restored and tumor growth reduced in mice with Treg-specific glucocorticoid receptor deficiency. Importantly, pharmacologic inhibition of 11ß-HSD1 reduced tumor growth to the same degree as gene knockout and rendered immunotherapy-resistant tumors susceptible to PD-1 blockade. Given that HSD11B1 expression is upregulated in many human tumors and that inhibition of 11ß-HSD1 is well tolerated in clinical studies, these data suggest that targeting 11ß-HSD1 may be a beneficial adjunct in cancer therapy.

Effects of sex steroids on thymic epithelium and thymocyte development.

Sex steroid hormones have major effects on the thymus. Age-related increases in androgens and estrogens and pregnancy-induced increases in progestins all cause dramatic thymic atrophy. Atrophy can also be induced by treatment with exogenous sex steroids and reversed by ablation of endogenous sex steroids. Although these observations are frequently touted as evidence of steroid lymphotoxicity, they are often driven by steroid signaling in thymic epithelial cells (TEC), which are highly steroid responsive. Here, we outline the effects of sex steroids on the thymus and T cell development. We focus on studies that have examined steroid signaling in vivo, aiming to emphasize the actions of endogenous steroids which, via TEC, have remarkable programming effects on the TCR repertoire. Due to the dramatic effects of steroids on TEC, especially thymic involution, the direct effects of sex steroid signaling in thymocytes are less well understood. We outline studies that could be important in addressing these possibilities, and highlight suggestive findings of sex steroid generation within the thymus itself.

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.

Glucocorticoids in T cell development, differentiation and function.

Glucocorticoids (GCs) are small lipid hormones produced by the adrenals that maintain organismal homeostasis. Circadian and stress-induced changes in systemic GC levels regulate metabolism, cardiovascular and neural function, reproduction and immune activity. Our understanding of GC effects on immunity comes largely from administration of exogenous GCs to treat immune or inflammatory disorders. However, it is increasingly clear that endogenous GCs both promote and suppress T cell immunity. Examples include selecting an appropriate repertoire of T cell receptor (TCR) self-affinities in the thymus, regulating T cell trafficking between anatomical compartments, suppressing type 1 T helper (TH1) cell responses while permitting TH2 cell and, especially, IL-17-producing T helper cell responses, and promoting memory T cell differentiation and maintenance. Furthermore, in addition to functioning at a distance, extra-adrenal (local) production allows GCs to act as paracrine signals, specifically targeting activated T cells in various contexts in the thymus, mucosa and tumours. These pleiotropic effects on different T cell populations during development and immune responses provide a nuanced understanding of how GCs shape immunity.

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.

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.

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.

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.

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.

Discovery and Characterization of a Biologically Active Non-ATP-Competitive p38 MAP Kinase Inhibitor.

Mitogen-activated protein kinase (MAPK) p38 is part of a broad and ubiquitously expressed family of MAPKs whose activity is responsible for mediating an intracellular response to extracellular stimuli through a phosphorylation cascade. p38 is central to this signaling node and is activated by upstream kinases while being responsible for activating downstream kinases and transcription factors via phosphorylation. Dysregulated p38 activity is associated with numerous autoimmune disorders and has been implicated in the progression of several types of cancer. A number of p38 inhibitors have been tested in clinical trials, with none receiving regulatory approval. One characteristic shared by all of the compounds that failed clinical trials is that they are all adenosine triphosphate (ATP)-competitive p38 inhibitors. Seeing this lack of mechanistic diversity as an opportunity, we screened ~32,000 substances in search of novel p38 inhibitors. Among the inhibitors discovered is a compound that is both non-ATP competitive and biologically active in cell-based models for p38 activity. This is the first reported discovery of a non-ATP-competitive p38 inhibitor that is active in cells and, as such, may enable new pharmacophore designs for both therapeutic and basic research to better understand and exploit non-ATP-competitive inhibitors of p38 activity.

Selective inhibition of the p38 alternative activation pathway in infiltrating T cells inhibits pancreatic cancer progression.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive neoplasm characterized by a marked fibro-inflammatory microenvironment, the presence of which can promote both cancer induction and growth. Therefore, selective manipulation of local cytokines is an attractive, although unrealized, therapeutic approach. T cells possess a unique mechanism of p38 mitogen-activated protein kinase (MAPK) activation downstream of T cell receptor (TCR) engagement through the phosphorylation of Tyr323 (pY323). This alternative p38 activation pathway is required for pro-inflammatory cytokine production. Here we show in human PDAC that a high percentage of infiltrating pY323(+) T cells was associated with large numbers of tumor necrosis factor (TNF)-α- and interleukin (IL)-17-producing CD4(+) tumor-infiltrating lymphocytes (TILs) and aggressive disease. The growth of mouse pancreatic tumors was inhibited by genetic ablation of the alternative p38 pathway, and transfer of wild-type CD4(+) T cells, but not those lacking the alternative pathway, enhanced tumor growth in T cell-deficient mice. Notably, a plasma membrane-permeable peptide derived from GADD45-α, the naturally occurring inhibitor of p38 pY323(+) (ref. 7), reduced CD4(+) TIL production of TNF-α, IL-17A, IL-10 and secondary cytokines, halted growth of implanted tumors and inhibited progression of spontaneous KRAS-driven adenocarcinoma in mice. Thus, TCR-mediated activation of CD4(+) TILs results in alternative p38 activation and production of protumorigenic factors and can be targeted for therapeutic benefit.

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.

Ionizing Radiation Impairs T Cell Activation by Affecting Metabolic Reprogramming.

Ionizing radiation has a variety of acute and long-lasting adverse effects on the immune system. Whereas measureable effects of radiation on immune cell cytotoxicity and population change have been well studied in human and animal models, little is known about the functional alterations of the surviving immune cells after ionizing radiation. The objective of this study was to delineate the effects of radiation on T cell function by studying the alterations of T cell receptor activation and metabolic changes in activated T cells isolated from previously irradiated animals. Using a global metabolomics profiling approach, for the first time we demonstrate that ionizing radiation impairs metabolic reprogramming of T cell activation, which leads to substantial decreases in the efficiency of key metabolic processes required for activation, such as glucose uptake, glycolysis, and energy metabolism. In-depth understanding of how radiation impacts T cell function highlighting modulation of metabolism during activation is not only a novel approach to investigate the pivotal processes in the shift of T cell homeostasis after radiation, it also may lead to new targets for therapeutic manipulation in the combination of radiotherapy and immune therapy. Given that appreciable effects were observed with as low as 10 cGy, our results also have implications for low dose environmental exposures.

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.