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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.

Wang et al. show cooperation between MYD88L265P and CD79B mutations dysregulating B cell responses to self-antigen and differentiation into plasma cells. Their results reveal that CD79B and surface IgM constitute a rate-limiting checkpoint against MYD88L265P, explaining the co-occurrence of MYD88 and CD79B mutations in human lymphomas., 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.

Background Patients frequently report antibiotic allergies; however, only 10% of labelled patients have a true allergy. Aim We investigated the documentation of antibiotic ‘allergy’ labels (AAL) and the effect of labelling on clinical outcomes, in a West Australian adult tertiary hospital. Methods Retrospective cross-sectional analysis of patients captured in the 2013 and 2014 National Antimicrobial Prescribing Surveys was carried out. Data were collected on documented antibiotic adverse drug reactions, antibiotic cost, prescribing appropriateness, prevalence of multi-drug resistant organisms, length of stay, intensive care admission and readmissions. Results Of the 687 patients surveyed, 278 (40%) were aged 70 or above, 365 (53%) were male and 279 (41%) were prescribed antibiotics. AAL were recorded in 122 (18%) patients and the majority were penicillin labels (n = 87; 71%). Details of AAL were documented for 80 of 141 (57%) individual allergy labels, with 61 describing allergic symptoms. Patients with beta-lactam allergy labels received fewer penicillins (P = 0.0002) and more aminoglycosides (P = 0.043) and metronidazole (P = 0.021) than patients without beta-lactam labels. Five patients received an antibiotic that was contraindicated according to their allergy status. Patients with AAL had significantly more hospital readmissions within 4 weeks (P = 0.001) and 6 months (P = 0.025) of discharge, compared with unlabelled patients. The majority (81%) of readmitted labelled patients had major infections. Conclusions AAL are common, but poorly documented in hospital records. Patients with AAL are significantly more likely to require alternative antibiotics and hospital readmissions. There may be a role for antibiotic allergy delabelling to mitigate the clinical and economic burdens for patients with invalid allergy labels.

Resisting or 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 series of missense variants in the immune response inhibitor A20 (encoded byTNFAIP3), substituting non-catalytic residues of the ubiquitin protease domain to diminish IκB-dependent phosphorylation and activation of A20. Two A20 variants with partial phosphorylation deficits appeared beneficial: one originating in Denisovans and introgressed in modern humans throughout Oceania, and another in a mouse strain resistant to Coxsackievirus. By contrast, a variant with 95% loss of phosphorylation caused spontaneous inflammatory disease in humans and mice. Analysis of the partial phosphorylation variant in mice revealed diminished tolerance of bacterial lipopolysaccharide or to poxvirus inoculation as trade-offs for enhanced immunity.One Sentence SummaryModern and ancient variants reveal a genetically tunable element for balancing immunity and microbial tolerance.

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.

B cells expressing the MYD88 L265P mutation undergo rapid TLR ligand-independent proliferation that is self-limiting unless apoptosis is opposed., MYD88L265P 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 MYD88L265P. The mutation induced rapid B cell division in the absence of exogenous TLR ligands and was inhibited by Unc93b13d mutation and chloroquine or TLR9 deficiency, indicating continued dependence on upstream TLR9 activation. Proliferation and NF-κB activation induced by MYD88L265P were nevertheless rapidly countered by the induction of TNFAIP3, an NF-κB inhibitor frequently inactivated in MYD88L265P–bearing lymphomas, and extinguished by Bim-dependent apoptosis. MYD88L265P 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.

CARD11 signaling determines whether antigen stimulation induces B cells to proliferate or die., 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.

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.

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.

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 (PAMPs) 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 haematological 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 (ABC-DLBCL) and almost 100% of Waldenström’s macroglobulinemia (WM) further highlight the involvement of TLRs in these malignancies. MYD88 mutations result in the chronic activation of TLR signalling 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 Toll-like receptors function and signalling in normal or inflammatory conditions, and how mutations can also hijack the TLR signalling pathways to give rise to cancer. Lastly, we discuss how potential therapeutic agents could be used to restore normal responses to TLRs and have long lasting anti-tumour effects.

Mice lacking activity of the intramembrane protease SPPL2A exhibit humoral immunodeficiency and lack mature B cell subsets., 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.

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 athttps://github.com/mattmattmattmatt/DeepSNVMiner.

Introduction: Diffuse large B-cell lymphoma (DLBCL) is an aggressive lymphoma with ~30% of cases failing treatment or relapsing after R-CHOP chemotherapy. Treatment options for refractory/ relapsed DLBCL are limited and often have a poor outcome. Tumour evolution and selection of a chemo resistant clone are likely explanations making it important to understand clonal heterogeneity at diagnosis and relapse. Green et al have noted a hierarchical acquisition of mutations with disease progression in follicular lymphoma (FL), with BCL2 and CREBBP mutations seen in earlier precursors and MLL2 and TNFRSF14 mutations proposed to be later events. Notably, 1 case analysed at diagnosis and at relapse had variable IgVH indicating origin in a more immature precursors. [Green, M.R., Blood, 2013]. More recent studies indicate that the dominant clone of FL and transformed FL arise by either convergent or divergent evolution from a common mutated precursor through the acquisition of additional genetic events [Pasqualucci, L., Cell Rep, 2014]. Defining the deregulated pathways and oncogenic mutations at diagnosis and later at relapse could help identify novel therapeutic targets for treatment at relapse. We aimed to map clonal evolution at relapse using immunophenotyping, sequencing for the Immunoglobulin variable heavy chain gene (IGHV) and genotyping for known somatic mutations in key lymphoma genes, within matched diagnostic and relapsed DLBCL pairs. Methods: Six matched diagnostic and relapsed DLBCL cryopreserved samples were subjected to comprehensive immunophenotyping using 10-colour B-cell flow cytometry panels based on normal ontogeny. PCR on initial and relapsed samples was performed to amplify clonally rearranged IgHV genes from fresh/paraffin embedded primary diagnostic samples. Sanger sequencing was done for IgHV mutation status by targeting the conserved framework regions (FR) FR1 [IGHA: VHFR1-JH] or FR3 [IGHC: VHFR3-JH] using the Invivoscribe kit based on the Biomed-2 protocols. A custom Haloplex library preparation kit designed and ordered from Agilent was used to capture the exons of ~40 genes commonly mutated in DLBCL. Targeted fragments were PCR amplified and target enriched samples sequenced on an Illumina MiSeq sequencer Results: Table 1. Immunophenotypic diversity was noted among cases but the phenotype did not change at relapse. IGHV sequencing showed that 3 cases with two distinct lymphoma clones at diagnosis evolved to a single detectable IGHV clonal population at relapse. Lymphoma-associated somatic mutations detected at diagnosis were preserved at relapse and in 4 cases, acquisition of additional mutations was observed. Table 1: Immunophenotype, IgHV sequence and somatic lymphoma mutations in paired DLBCL samples at diagnosis and relapse Patient number Disease course Immunophenotype IgHV sequence Lymphoma mutations DL157 Diagnosis IgM+, IgD+++, IgG-, CD27-, CD21+++, CD38++, CD10- N/A (not available) EP300 I->V EZH2 D->H NOTCH2 L->H TRAF2 A->T DL157 Relapse Same as diagnosis IgHV3-64 IgHV3-30 EZH2 D->H FOXO1G->D NOTCH2L->HTRAF2 A->T DL214 Diagnosis IgM+++,IgD++, IgG-, CD27-, CD21-, CD38+++, CD10+ IgHV4-30 IgHV4-39 N/A DL214 Relapse Same as diagnosis IgHV4-39 EP300 I->V MYD88 L->P NOTCH1 R->W PRDM1 C->Y SPEN D->E TP53 Y->N DL215 Diagnosis IgM-,IgD- IgG++, CD27++,CD21++, CD38-, CD10- IGHV3-23 IGHV3-23D KMT2D R->C MYD88 L->P NOTCH1 E->K DL215 Relapse Same as diagnosis IGHV3-23 BTG1 H->Y BTG1 Q->Stop BTG2 S->N FOXO1 V->E GNA13 R->H KMT2D R->C MYD88 L->P NOTCH1 E->K PIM1 L->F PIM1 K->N PIM1 V->L DL245 Diagnosis N/A IGHV3-11 IGHV3-33 EP300,I->V,997 NOTCH2,L->V,1559 DL245 Relapse N/A IGHV3-11 EP300,I->V,997 NOTCH2,L->V,1559 DL213 Diagnosis IgM+,IgD-, IgG-, CD27-,CD21+++, CD38+++, CD10++ IGHV3-15 ETS1 A->T EZH2 D->H PRDM1 L->F TNFRSF14 C->S DL213 Relapse Same as diagnosis IGHV3-15 ETS1 A->T EZH2 D->H KMT2D R->Stop PRDM1 L->FSTAT3 R->W DL252 Diagnosis IgM-,IgD++, IgG-, CD27-,CD21-, CD38+, CD10- IGHV1-2 NOTCH2,P->A,2359 TNFRSF14,V->I,241 DL252 Relapse Same as diagnosis IGHV1-2 N/A Conclusions: While multiple IGHV sub-clones are present at diagnosis of DLBCL, evolution of a single clone is noted at relapse (convergent model) with acquisition of additional somatic mutations. Immunophenotyping using B-cell maturation flow cytometry panels demonstrates heterogeneity of differentiation between cases but no change in immunophenotype at relapse. Disclosures No relevant conflicts of interest to declare.

Correspondence: Yogesh S Jeelall Immunogenomics Laboratory, Department of Immunology, The John Curtin School of Medical Research, The Australian National University, Building 131, Garran Road, Acton, ACT 0200, Australia Tel +612 6125 1393 Fax +612 6125 2595 Email yogesh.jeelall@anu.edu.au Abstract: B cell neoplasms co-opt the molecular machinery of normal B cells for their survival. Technological advances in cancer genomics has significantly contributed to uncovering the root cause of aggressive lymphomas, revealing a previously unknown link between TLR signaling and B cell neoplasm. Recurrent oncogenic mutations in MYD88 have been found in 39% of the activated B cell-like subtype of diffuse large B cell lymphoma (ABC DLBCL). Interestingly, 29% of ABC DLBCL have a single amino acid substitution of proline for the leucine at position 265 (L265P), and the exact same variant has also been identified in a number of lymphoid malignancies. The MYD88 L265P variant was recently identified in 90% of Wadenstrom’s macroglobulinemia patients. These recent developments warrant the need for novel diagnostic tools as well as targeted therapeutics. In this review, we discuss the physiological functions of MYD88 and focus on its role in B cell lymphomas, evaluating the potential for targeting oncogenic MYD88 in lymphoma.