The alarmin interleukin-33 promotes the expansion and preserves the stemness of Tcf-1+ CD8+ T cells in chronic viral infection.

T cell factor 1 (Tcf-1) expressing CD8+ T cells exhibit stem-like self-renewing capacity, rendering them key for immune defense against chronic viral infection and cancer. Yet, the signals that promote the formation and maintenance of these stem-like CD8+ T cells (CD8+SL) remain poorly defined. Studying CD8+ T cell differentiation in mice with chronic viral infection, we identified the alarmin interleukin-33 (IL-33) as pivotal for the expansion and stem-like functioning of CD8+SL as well as for virus control. IL-33 receptor (ST2)-deficient CD8+ T cells exhibited biased end differentiation and premature loss of Tcf-1. ST2-deficient CD8+SL responses were restored by blockade of type I interferon signaling, suggesting that IL-33 balances IFN-I effects to control CD8+SL formation in chronic infection. IL-33 signals broadly augmented chromatin accessibility in CD8+SL and determined these cells' re-expansion potential. Our study identifies the IL-33-ST2 axis as an important CD8+SL-promoting pathway in the context of chronic viral infection.

Central tolerance shapes the neutralizing B cell repertoire against a persisting virus in its natural host.

Viral mimicry of host cell structures has been postulated to curtail the B cell receptor (BCR) repertoire against persisting viruses through tolerance mechanisms. This concept awaits, however, experimental testing in a setting of natural virus-host relationship. We engineered mouse models expressing a monoclonal BCR specific for the envelope glycoprotein of lymphocytic choriomeningitis virus (LCMV), a naturally persisting mouse pathogen. When the heavy chain of the LCMV-neutralizing antibody KL25 was paired with its unmutated ancestor light chain, most B cells underwent receptor editing, a behavior reminiscent of autoreactive clones. In contrast, monoclonal B cells expressing the same heavy chain in conjunction with the hypermutated KL25 light chain did not undergo receptor editing but exhibited low levels of surface IgM, suggesting that light chain hypermutation had lessened KL25 autoreactivity. Upon viral challenge, these IgMlow cells were not anergic but up-regulated IgM, participated in germinal center reactions, produced antiviral antibodies, and underwent immunoglobulin class switch as well as further affinity maturation. These studies on a persisting virus in its natural host species suggest that central tolerance mechanisms prune the protective antiviral B cell repertoire.

Vaccine-elicited CD4 T cells prevent the deletion of antiviral B cells in chronic infection.

Chronic viral infections subvert protective B cell immunity. An early type I interferon (IFN-I)-driven bias to short-lived plasmablast differentiation leads to clonal deletion, so-called "decimation," of antiviral memory B cells. Therefore, prophylactic countermeasures against decimation remain an unmet need. We show that vaccination-induced CD4 T cells prevented the decimation of naïve and memory B cells in chronically lymphocytic choriomeningitis virus (LCMV)-infected mice. Although these B cell responses were largely T independent when IFN-I was blocked, preexisting T help assured their sustainability under conditions of IFN-I-driven inflammation by instructing a germinal center B cell transcriptional program. Prevention of decimation depended on T cell-intrinsic Bcl6 and Tfh progeny formation. Antigen presentation by B cells, interactions with antigen-specific T helper cells, and costimulation by CD40 and ICOS were also required. Importantly, B cell-mediated virus control averted Th1-driven immunopathology in LCMV-challenged animals with preexisting CD4 T cell immunity. Our findings show that vaccination-induced Tfh cells represent a cornerstone of effective B cell immunity to chronic virus challenge, pointing the way toward more effective B cell-based vaccination against persistent viral diseases.

Activation of IGF1R/p110ß/AKT/mTOR confers resistance to α-specific PI3K inhibition.

BACKGROUND: The PI3K pathway is hyperactivated in many cancers, including 70 % of breast cancers. Pan- and isoform-specific inhibitors of the PI3K pathway are currently being evaluated in clinical trials. However, the clinical responses to PI3K inhibitors when used as single agents are not as efficient as expected. METHODS: In order to anticipate potential molecular mechanisms of resistance to the p110α isoform-selective inhibitor BYL719, we developed resistant breast cancer cell lines, assessed the concomitant changes in cellular signaling pathways using unbiased phosphotyrosine proteomics and characterized the mechanism of resistance using pharmacological inhibitors. RESULTS: We found an increase in IGF1R, IRS1/IRS2 and p85 phosphorylation in the resistant lines. Co-immunoprecipitation experiments identified an IGF1R/IRS/p85/p110ß complex that causes the activation of AKT/mTOR/S6K and stifles the effects of BYL719. Pharmacological inhibition of members of this complex reduced mTOR/S6K activation and restored sensitivity to BYL719. CONCLUSION: Our study demonstrates that the IGF1R/p110ß/AKT/mTOR axis confers resistance to BYL719 in PIK3CA mutant breast cancers. This provides a rationale for the combined targeting of p110α with IGF1R or p110ß in patients with breast tumors harboring PIK3CA mutations.

AMPK promotes skeletal muscle autophagy through activation of forkhead FoxO3a and interaction with Ulk1.

In skeletal muscle, protein levels are determined by relative rates of protein synthesis and breakdown. The balance between synthesis and degradation of intracellular components determines the overall muscle fiber size. AMP-activated protein kinase (AMPK), a sensor of cellular energy status, was recently shown to increase myofibrillar protein degradation through the expression of MAFbx and MuRF1. In the present study, the effect of AMPK activation by AICAR on autophagy was investigated in muscle cells. Our results show that FoxO3a transcription factor activation by AMPK induces the expression of the autophagy-related proteins LC3B-II, Gabarapl1, and Beclin1 in primary mouse skeletal muscle myotubes and in the Tibialis anterior (TA) muscle. Time course studies reveal that AMPK activation by AICAR leads to a transient nuclear relocalization of FoxO3a followed by an increase of its cytosolic level. Moreover, AMPK activation leads to the inhibition of mTORC1 and its subsequent dissociation of Ulk1, Atg13, and FIP200 complex. Interestingly, we identify Ulk1 as a new interacting partner of AMPK in muscle cells and we show that Ulk1 is associated with AMPK under normal conditions and dissociates from AMPK during autophagy process. Moreover, we find that AMPK phosphorylates FoxO3a and Ulk1. In conclusion, our data show that AMPK activation stimulates autophagy in skeletal muscle cells through its effects on the transcriptional function of FoxO3a and takes part in the initiation of autophagosome formation by interacting with Ulk1. Here, we present new evidences that AMPK plays a crucial role in the fine tuning of protein expression programs that control skeletal muscle mass.

Chronic Viral Infection Promotes Efficient Germinal Center B Cell Responses.

Persistent viral infections subvert key elements of adaptive immunity. To compare germinal center (GC) B cell responses in chronic and acute lymphocytic choriomeningitis virus infection, we exploit activation-induced deaminase (AID) fate-reporter mice and perform adoptive B cell transfer experiments. Chronic infection yields GC B cell responses of higher cellularity than acute infections do, higher memory B cell and antibody secreting cell output for longer periods of time, a better representation of the late B cell repertoire in serum immunoglobulin, and higher titers of protective neutralizing antibodies. GC B cells of chronically infected mice are similarly hypermutated as those emerging from acute infection. They efficiently adapt to viral escape variants and even in hypermutation-impaired AID mutant mice, chronic infection selects for GC B cells with hypermutated B cell receptors (BCRs) and neutralizing antibody formation. These findings demonstrate that, unlike for CD8+ T cells, chronic viral infection drives a functional, productive, and protective GC B cell response.

Reversible optic neuropathy with OPA1 exon 5b mutation.

A new c.740G>A (R247H) mutation in OPA1 alternate spliced exon 5b was found in a patient presenting with bilateral optic neuropathy followed by partial, spontaneous visual recovery. R247H fibroblasts from the patient and his unaffected father presented unusual highly tubular mitochondrial network, significant increased susceptibility to apoptosis, oxidative phosphorylation uncoupling, and altered OPA1 protein profile, supporting the pathogenicity of this mutation. These results suggest that the clinical spectrum of the OPA1-associated optic neuropathies may be larger than previously described, and that spontaneous recovery may occur in cases harboring an exon 5b mutation.

Interferon-driven deletion of antiviral B cells at the onset of chronic infection.

Inadequate antibody responses and perturbed B cell compartments represent hallmarks of persistent microbial infections, but the mechanisms whereby persisting pathogens suppress humoral immunity remain poorly defined. Using adoptive transfer experiments in the context of a chronic lymphocytic choriomeningitis virus (LCMV) infection of mice, we have documented rapid depletion of virus-specific B cells that coincided with the early type I interferon response to infection. We found that the loss of activated B cells was driven by type I interferon (IFN-I) signaling to several cell types including dendritic cells, T cells and myeloid cells. Intriguingly, this process was independent of B cell-intrinsic IFN-I sensing and resulted from biased differentiation of naïve B cells into short-lived antibody-secreting cells. The ability to generate robust B cell responses was restored upon IFN-I receptor blockade or, partially, when experimentally depleting myeloid cells or the IFN-I-induced cytokines interleukin 10 and tumor necrosis factor alpha. We have termed this IFN-I-driven depletion of B cells "B cell decimation". Strategies to counter "B cell decimation" should thus help us better leverage humoral immunity in the combat against persistent microbial diseases.

VEGF-mediated angiogenesis links EMT-induced cancer stemness to tumor initiation.

An epithelial-mesenchymal transition (EMT) underlies malignant tumor progression and metastatic spread by enabling cancer cells to depart from the primary tumor, invade surrounding tissue, and disseminate to distant organs. EMT also enriches for cancer stem cells (CSC) and increases the capacity of cancer cells to initiate and propagate tumors upon transplantation into immune-deficient mice, a major hallmark of CSCs. However, the molecular mechanisms promoting the tumorigenicity of cancer cells undergoing an EMT and of CSCs have remained widely elusive. We here report that EMT confers efficient tumorigenicity to murine breast cancer cells by the upregulated expression of the proangiogenic factor VEGF-A and by increased tumor angiogenesis. On the basis of these data, we propose a novel interpretation of the features of CSCs with EMT-induced, VEGF-A-mediated angiogenesis as the connecting mechanism between cancer cell stemness and tumor initiation.

The translation regulatory subunit eIF3f controls the kinase-dependent mTOR signaling required for muscle differentiation and hypertrophy in mouse.

The mTORC1 pathway is required for both the terminal muscle differentiation and hypertrophy by controlling the mammalian translational machinery via phosphorylation of S6K1 and 4E-BP1. mTOR and S6K1 are connected by interacting with the eIF3 initiation complex. The regulatory subunit eIF3f plays a major role in muscle hypertrophy and is a key target that accounts for MAFbx function during atrophy. Here we present evidence that in MAFbx-induced atrophy the degradation of eIF3f suppresses S6K1 activation by mTOR, whereas an eIF3f mutant insensitive to MAFbx polyubiquitination maintained persistent phosphorylation of S6K1 and rpS6. During terminal muscle differentiation a conserved TOS motif in eIF3f connects mTOR/raptor complex, which phosphorylates S6K1 and regulates downstream effectors of mTOR and Cap-dependent translation initiation. Thus eIF3f plays a major role for proper activity of mTORC1 to regulate skeletal muscle size.

MAFbx/Atrogin-1 controls the activity of the initiation factor eIF3-f in skeletal muscle atrophy by targeting multiple C-terminal lysines.

We recently presented evidence that the subunit eIF3-f of the eukaryotic initiation translation factor eIF3 that interacts with the E3-ligase Atrogin-1/muscle atrophy F-box (MAFbx) for polyubiquitination and proteasome-mediated degradation is a key target that accounts for MAFbx function during muscle atrophy. To understand this process, deletion analysis was used to identify the region of eIF3-f that is required for its proteolysis. Here, we report that the highly conserved C-terminal domain of eIF3-f is implicated for MAFbx-directed polyubiquitination and proteasomal degradation. Site-directed mutagenesis of eIF3-f revealed that the six lysine residues within this domain are required for full polyubiquitination and degradation by the proteasome. In addition, mutation of these six lysines (mutant K(5-10)R) displayed hypertrophic activity in cellulo and in vivo and was able to protect against starvation-induced muscle atrophy. Taken together, our data demonstrate that the C-terminal modifications, believed to be critical for proper eIF3-f regulation, are essential and contribute to a fine-tuning mechanism that plays an important role for eIF3-f function in skeletal muscle.

Inhibition of atrogin-1/MAFbx mediated MyoD proteolysis prevents skeletal muscle atrophy in vivo.

Ubiquitin ligase Atrogin1/Muscle Atrophy F-box (MAFbx) up-regulation is required for skeletal muscle atrophy but substrates and function during the atrophic process are poorly known. The transcription factor MyoD controls myogenic stem cell function and differentiation, and seems necessary to maintain the differentiated phenotype of adult fast skeletal muscle fibres. We previously showed that MAFbx mediates MyoD proteolysis in vitro. Here we present evidence that MAFbx targets MyoD for degradation in several models of skeletal muscle atrophy. In cultured myotubes undergoing atrophy, MAFbx expression increases, leading to a cytoplasmic-nuclear shuttling of MAFbx and a selective suppression of MyoD. Conversely, transfection of myotubes with sh-RNA-mediated MAFbx gene silencing (shRNAi) inhibited MyoD proteolysis linked to atrophy. Furthermore, overexpression of a mutant MyoDK133R lacking MAFbx-mediated ubiquitination prevents atrophy of mouse primary myotubes and skeletal muscle fibres in vivo. Regarding the complex role of MyoD in adult skeletal muscle plasticity and homeostasis, its rapid suppression by MAFbx seems to be a major event leading to skeletal muscle wasting. Our results point out MyoD as the second MAFbx skeletal muscle target by which powerful therapies could be developed.