Denisovan, modern human and mouse TNFAIP3 alleles tune A20 phosphorylation and immunity.

Resisting and tolerating microbes are alternative strategies to survive infection, but little is known about the evolutionary mechanisms controlling this balance. Here genomic analyses of anatomically modern humans, extinct Denisovan hominins and mice revealed a TNFAIP3 allelic series with alterations in the encoded immune response inhibitor A20. Each TNFAIP3 allele encoded substitutions at non-catalytic residues of the ubiquitin protease OTU domain that diminished IκB kinase-dependent phosphorylation and activation of A20. Two TNFAIP3 alleles encoding A20 proteins with partial phosphorylation deficits seemed to be beneficial by increasing immunity without causing spontaneous inflammatory disease: A20 T108A;I207L, originating in Denisovans and introgressed in modern humans throughout Oceania, and A20 I325N, from an N-ethyl-N-nitrosourea (ENU)-mutagenized mouse strain. By contrast, a rare human TNFAIP3 allele encoding an A20 protein with 95% loss of phosphorylation, C243Y, caused spontaneous inflammatory disease in humans and mice. Analysis of the partial-phosphorylation A20 I325N allele in mice revealed diminished tolerance of bacterial lipopolysaccharide and poxvirus inoculation as tradeoffs for enhanced immunity.

Omenn syndrome associated with a functional reversion due to a somatic second-site mutation in CARD11 deficiency.

Omenn syndrome (OS) is a severe immunodeficiency associated with erythroderma, lymphoproliferation, elevated IgE, and hyperactive oligoclonal T cells. A restricted T-cell repertoire caused by defective thymic T-cell development and selection, lymphopenia with homeostatic proliferation, and lack of regulatory T cells are considered key factors in OS pathogenesis. We report 2 siblings presenting with cytomegalovirus (CMV) and Pneumocystis jirovecii infections and recurrent sepsis; one developed all clinical features of OS. Both carried homozygous germline mutations in CARD11 (p.Cys150*), impairing NF-κB signaling and IL-2 production. A somatic second-site mutation reverting the stop codon to a missense mutation (p.Cys150Leu) was detected in tissue-infiltrating T cells of the OS patient. Expression of p.Cys150Leu in CARD11-deficient T cells largely reconstituted NF-κB signaling. The reversion likely occurred in a prethymic T-cell precursor, leading to a chimeric T-cell repertoire. We speculate that in our patient the functional advantage of the revertant T cells in the context of persistent CMV infection, combined with lack of regulatory T cells, may have been sufficient to favor OS. This first observation of OS in a patient with a T-cell activation defect suggests that severely defective T-cell development or homeostatic proliferation in a lymphopenic environment are not required for this severe immunopathology.

CARD11 gain-of-function mutation drives cell-autonomous accumulation of PD-1+ ICOShigh activated T cells, T-follicular, T-regulatory and T-follicular regulatory cells.

Introduction: Germline CARD11 gain-of-function (GOF) mutations cause B cell Expansion with NF-κB and T cell Anergy (BENTA) disease, whilst somatic GOF CARD11 mutations recur in diffuse large B cell lymphoma (DLBCL) and in up to 30% of the peripheral T cell lymphomas (PTCL) adult T cell leukemia/lymphoma (ATL), cutaneous T cell lymphoma (CTCL) and Sezary Syndrome. Despite their frequent acquisition by PTCL, the T cell-intrinsic effects of CARD11 GOF mutations are poorly understood. Methods: Here, we studied B and T lymphocytes in mice with a germline Nethyl-N-nitrosourea (ENU)-induced Card11M365K mutation identical to a mutation identified in DLBCL and modifying a conserved region of the CARD11 coiled-coil domain recurrently mutated in DLBCL and PTCL. Results and discussion: Our results demonstrate that CARD11.M365K is a GOF protein that increases B and T lymphocyte activation and proliferation following antigen receptor stimulation. Germline Card11M365K mutation was insufficient alone to cause B or T-lymphoma, but increased accumulation of germinal center (GC) B cells in unimmunized and immunized mice. Card11M365K mutation caused cell-intrinsic over-accumulation of activated T cells, T regulatory (TREG), T follicular (TFH) and T follicular regulatory (TFR) cells expressing increased levels of ICOS, CTLA-4 and PD-1 checkpoint molecules. Our results reveal CARD11 as an important, cell-autonomous positive regulator of TFH, TREG and TFR cells. They highlight T cell-intrinsic effects of a GOF mutation in the CARD11 gene, which is recurrently mutated in T cell malignancies that are often aggressive and associated with variable clinical outcomes.

Effects of localized neurotrophin gene expression on spiral ganglion neuron resprouting in the deafened cochlea.

A cochlear implant may be used to electrically stimulate spiral ganglion neurons (SGNs) in people with severe sensorineural hearing loss (SNHL). However, these neurons progressively degenerate after SNHL due to loss of neurotrophins normally supplied by sensory hair cells (HCs). Experimentally, exogenous neurotrophin administration prevents SGN degeneration but can also result in abnormal resprouting of their peripheral fibers. This study aimed to create a target-derived neurotrophin source to increase neuron survival and redirect fiber resprouting following SNHL. Adenoviral (Ad) vectors expressing green fluorescent protein (GFP) alone or in combination with brain-derived neurotrophic factor (BDNF) or neurotrophin-3 (NT3) were injected into the cochlear scala tympani or scala media of guinea-pigs (GPs) deafened via aminoglycosides for 1 week. After 3 weeks, cochleae were examined for gene expression, neuron survival, and the projection of peripheral fibers in response to gene expression. Injection of vectors into the scala media resulted in more localized gene expression than scala tympani injection with gene expression consistently observed within the partially degenerated organ of Corti. There was also greater neuron survival and evidence of localized fiber responses to neurotrophin-expressing cells within the organ of Corti from scala media injections (P < 0.05), a first step in promoting organized resprouting of auditory peripheral fibers via gene therapy.

The role of immunological testing and intervention in reproductive medicine: A fertile collaboration?

Advances in reproductive medicine have significantly increased the success of fertility treatments. Nevertheless, some women experience recurrent implantation failure (RIF) after in-vitro fertilization (IVF) or recurrent pregnancy loss (RPL). Imbalances in the immune system and failure to achieve immune tolerance to the foetus have been implicated as potentially modifiable causes of idiopathic RIF and RPL. As such, women are increasingly being treated with immunomodulatory agents in an attempt to achieve a successful pregnancy. This systematic review examines the published evidence on immune changes in these patients, the use of immunomodulation therapies and diagnostic testing modalities to guide their use or to identify patient subsets most likely to benefit. The PubMed database was searched for the terms "recurrent implantation failure" and "recurrent pregnancy loss" in conjunction with T-helper (Th) cells and their subsets in particular; Th1, Th2, Th17 and T-regulatory (Treg) cells, natural killer (NK) cells, cytokine imbalance as well as immune modulators and immune suppressants. The reference lists of articles were examined to identify additional articles. There remains limited data on the immunological changes in cytokine and cellular profiles during the hormonal cycle as well as prior to, during and after implantation in health as well as idiopathic RIF and RPL. There is a need to advance immunological diagnostics to match the clinical need in this emerging field and to guide clinicians to make optimal and safe therapeutic choices. It is also imperative that the well-being of the infants conceived after such intervention is monitored.

Synergistic cooperation and crosstalk between MYD88L265P and mutations that dysregulate CD79B and surface IgM.

CD79B and MYD88 mutations are frequently and simultaneously detected in B cell malignancies. It is not known if these mutations cooperate or how crosstalk occurs. Here we analyze the consequences of CD79B and MYD88L265P mutations individually and combined in normal activated mouse B lymphocytes. CD79B mutations alone increased surface IgM but did not enhance B cell survival, proliferation, or altered NF-κB responsive markers. Conversely, B cells expressing MYD88L265P decreased surface IgM coupled with accumulation of endoglycosidase H-sensitive IgM intracellularly, resembling the trafficking block in anergic B cells repeatedly stimulated by self-antigen. Mutation or overexpression of CD79B counteracted the effect of MYD88L265P In B cells chronically stimulated by self-antigen, CD79B and MYD88L265P mutations in combination, but not individually, blocked peripheral deletion and triggered differentiation into autoantibody secreting plasmablasts. These results reveal that CD79B and surface IgM constitute a rate-limiting checkpoint against B cell dysregulation by MYD88L265P and provide an explanation for the co-occurrence of MYD88 and CD79B mutations in lymphomas.

DeepSNVMiner: a sequence analysis tool to detect emergent, rare mutations in subsets of cell populations.

Background. Massively parallel sequencing technology is being used to sequence highly diverse populations of DNA such as that derived from heterogeneous cell mixtures containing both wild-type and disease-related states. At the core of such molecule tagging techniques is the tagging and identification of sequence reads derived from individual input DNA molecules, which must be first computationally disambiguated to generate read groups sharing common sequence tags, with each read group representing a single input DNA molecule. This disambiguation typically generates huge numbers of reads groups, each of which requires additional variant detection analysis steps to be run specific to each read group, thus representing a significant computational challenge. While sequencing technologies for producing these data are approaching maturity, the lack of available computational tools for analysing such heterogeneous sequence data represents an obstacle to the widespread adoption of this technology. Results. Using synthetic data we successfully detect unique variants at dilution levels of 1 in a 1,000,000 molecules, and find DeeepSNVMiner obtains significantly lower false positive and false negative rates compared to popular variant callers GATK, SAMTools, FreeBayes and LoFreq, particularly as the variant concentration levels decrease. In a dilution series with genomic DNA from two cells lines, we find DeepSNVMiner identifies a known somatic variant when present at concentrations of only 1 in 1,000 molecules in the input material, the lowest concentration amongst all variant callers tested. Conclusions. Here we present DeepSNVMiner; a tool to disambiguate tagged sequence groups and robustly identify sequence variants specific to subsets of starting DNA molecules that may indicate the presence of a disease. DeepSNVMiner is an automated workflow of custom sequence analysis utilities and open source tools able to differentiate somatic DNA variants from artefactual sequence variants that likely arose during DNA amplification. The workflow remains flexible such that it may be customised to variants of the data production protocol used, and supports reproducible analysis through detailed logging and reporting of results. DeepSNVMiner is available for academic non-commercial research purposes at https://github.com/mattmattmattmatt/DeepSNVMiner.

Toll-Like Receptors and Cancer: MYD88 Mutation and Inflammation.

Pattern recognition receptors (PRRs) expressed on immune cells are crucial for the early detection of invading pathogens, in initiating early innate immune response and in orchestrating the adaptive immune response. PRRs are activated by specific pathogen-associated molecular patterns that are present in pathogenic microbes or nucleic acids of viruses or bacteria. However, inappropriate activation of these PRRs, such as the Toll-like receptors (TLRs), due to genetic lesions or chronic inflammation has been demonstrated to be a major cause of many hematological malignancies. Gain-of-function mutations in the TLR adaptor protein MYD88 found in 39% of the activated B cell type of diffuse large B cell lymphomas and almost 100% of Waldenström's macroglobulinemia further highlight the involvement of TLRs in these malignancies. MYD88 mutations result in the chronic activation of TLR signaling pathways, thus the constitutive activation of the transcription factor NFκB to promote cell survival and proliferation. These recent insights into TLR pathway driven malignancies warrant the need for a better understanding of TLRs in cancers and the development of novel anti-cancer therapies targeting TLRs. This review focuses on TLR function and signaling in normal or inflammatory conditions, and how mutations can hijack the TLR signaling pathways to give rise to cancer. Finally, we discuss how potential therapeutic agents could be used to restore normal responses to TLRs and have long lasting anti-tumor effects.

Consequences of the recurrent MYD88(L265P) somatic mutation for B cell tolerance.

MYD88(L265P) has recently been discovered as an extraordinarily frequent somatic mutation in benign monoclonal IgM gammopathy, Waldenström's macroglobulinemia, and diffuse large B cell lymphoma. In this study, we analyze the consequences for antigen-activated primary B cells of acquiring MYD88(L265P). The mutation induced rapid B cell division in the absence of exogenous TLR ligands and was inhibited by Unc93b1(3d) mutation and chloroquine or TLR9 deficiency, indicating continued dependence on upstream TLR9 activation. Proliferation and NF-κB activation induced by MYD88(L265P) were nevertheless rapidly countered by the induction of TNFAIP3, an NF-κB inhibitor frequently inactivated in MYD88(L265P)-bearing lymphomas, and extinguished by Bim-dependent apoptosis. MYD88(L265P) caused self-reactive B cells to accumulate in vivo only when apoptosis was opposed by Bcl2 overexpression. These results reveal checkpoints that fortify TLR responses against aberrant B cell proliferation in response to ubiquitous TLR and BCR self-ligands and suggest that tolerance failure requires the accumulation of multiple somatic mutations.

B cell survival, surface BCR and BAFFR expression, CD74 metabolism, and CD8- dendritic cells require the intramembrane endopeptidase SPPL2A.

Druggable proteins required for B lymphocyte survival and immune responses are an emerging source of new treatments for autoimmunity and lymphoid malignancy. In this study, we show that mice with an inactivating mutation in the intramembrane protease signal peptide peptidase-like 2A (SPPL2A) unexpectedly exhibit profound humoral immunodeficiency and lack mature B cell subsets, mirroring deficiency of the cytokine B cell-activating factor (BAFF). Accumulation of Sppl2a-deficient B cells was rescued by overexpression of the BAFF-induced survival protein B cell lymphoma 2 (BCL2) but not BAFF and was distinguished by low surface BAFF receptor and IgM and IgD B cell receptors. CD8-negative dendritic cells were also greatly decreased. SPPL2A deficiency blocked the proteolytic processing of CD74 MHC II invariant chain in both cell types, causing dramatic build-up of the p8 product of Cathepsin S and interfering with earlier steps in CD74 endosomal retention and processing. The findings illuminate an important role for the final step in the CD74-MHC II pathway and a new target for protease inhibitor treatment of B cell diseases.

Human lymphoma mutations reveal CARD11 as the switch between self-antigen-induced B cell death or proliferation and autoantibody production.

Self-tolerance and immunity are actively acquired in parallel through a poorly understood ability of antigen receptors to switch between signaling death or proliferation of antigen-binding lymphocytes in different contexts. It is not known whether this tolerance-immunity switch requires global rewiring of the signaling apparatus or if it can arise from a single molecular change. By introducing individual CARD11 mutations found in human lymphomas into antigen-activated mature B lymphocytes in mice, we find here that lymphoma-derived CARD11 mutations switch the effect of self-antigen from inducing B cell death into T cell-independent proliferation, Blimp1-mediated plasmablast differentiation, and autoantibody secretion. Our findings demonstrate that regulation of CARD11 signaling is a critical switch governing the decision between death and proliferation in antigen-stimulated mature B cells and that mutations in this switch represent a powerful initiator for aberrant B cell responses in vivo.