HSP110 sustains chronic NF-κB signaling in activated B-cell diffuse large B-cell lymphoma through MyD88 stabilization.

Activated B-cell diffuse large B-cell lymphoma (ABC-DLBCL) is an aggressive lymphoproliferative disorder involving chronic NF-κB activation. Several mutations in the BCR and MyD88 signaling pathway components, such as MyD88 L265P, are implicated in this aberrant activation. Among heat shock proteins, HSP110 has recently been identified as a prosurvival and/or proliferation factor in many cancers, but its role in ABC-DLBCL survival mechanisms remained to be established. We observed that short hairpin RNA-mediated HSP110 silencing decreased the survival of several ABC-DLBCL cell lines and decreased immunoglobulin M-MyD88 co-localization and subsequent NF-κB signaling. Conversely, overexpression of HSP110 in ABC-DLBCL or non-DLBCL cell lines increased NF-κB signaling, indicating a tight interplay between HSP110 and the NF-κB pathway. By using immunoprecipitation and proximity ligation assays, we identified an interaction between HSP110 and both wild-type MyD88 and MyD88 L265P. HSP110 stabilized both MyD88 forms with a stronger effect on MyD88 L265P, thus facilitating chronic NF-κB activation. Finally, HSP110 expression was higher in lymph node biopsies from patients with ABC-DLBCL than in normal reactive lymph nodes, and a strong correlation was found between the level of HSP110 and MyD88. In conclusion, we identified HSP110 as a regulator of NF-κB signaling through MyD88 stabilization in ABC-DLBCL. This finding reveals HSP110 as a new potential therapeutic target in ABC-DLBCL.

The fungal ligand chitin directly binds TLR2 and triggers inflammation dependent on oligomer size.

Chitin is the second most abundant polysaccharide in nature and linked to fungal infection and asthma. However, bona fide immune receptors directly binding chitin and signaling immune activation and inflammation have not been clearly identified because polymeric crude chitin with unknown purity and molecular composition has been used. By using defined chitin (N-acetyl-glucosamine) oligomers, we here identify six-subunit-long chitin chains as the smallest immunologically active motif and the innate immune receptor Toll-like receptor (TLR2) as a primary fungal chitin sensor on human and murine immune cells. Chitin oligomers directly bind TLR2 with nanomolar affinity, and this fungal TLR2 ligand shows overlapping and distinct signaling outcomes compared to known mycobacterial TLR2 ligands. Unexpectedly, chitin oligomers composed of five or less subunits are inactive, hinting to a size-dependent system of immuno-modulation that appears conserved in plants and humans. Since blocking of the chitin-TLR2 interaction effectively prevents chitin-mediated inflammation in vitro and in vivo, our study highlights the chitin-TLR2 interaction as a potential target for developing novel therapies in chitin-related pathologies and fungal disease.

Oncogenic MYD88 mutations in lymphoma: novel insights and therapeutic possibilities.

Oncogenic MYD88 mutations, most notably the Leu 265 Pro (L265P) mutation, were recently identified as potential driver mutations in various B-cell non-Hodgkin Lymphomas (NHLs). The L265P mutation is now thought to be common to virtually all NHLs and occurs in between 4 and 90% of cases, depending on the entity. Since it is tumor-specific, the mutation, and the pathways it regulates, might serve as advantageous therapeutic targets for both conventional chemotherapeutic intervention, as well as immunotherapeutic strategies. Here, we review recent progress on elucidating the molecular and cellular processes affected by the L265P mutation of MYD88, describe a new in vivo model for MyD88 L265P-mediated oncogenesis, and summarize how these findings could be exploited therapeutically by specific targeting of signaling pathways. In addition, we summarize current and explore future possibilities for conceivable immunotherapeutic approaches, such as L265P-derived peptide vaccination, adoptive transfer of L265P-restricted T cells, and use of T-cell receptor-engineered T cells. With clinical trials regarding their efficacy rapidly expanding to NHLs, we also discuss potential combinations of immune checkpoint inhibitors with the described targeted chemotherapies of L265P signaling networks, and/or with the above immunological approaches as potential ways of targeting MYD88-mutated lymphomas in the future.

B-cell-specific conditional expression of Myd88p.L252P leads to the development of diffuse large B-cell lymphoma in mice.

The adaptor protein MYD88 is critical for relaying activation of Toll-like receptor signaling to NF-κB activation. MYD88 mutations, particularly the p.L265P mutation, have been described in numerous distinct B-cell malignancies, including diffuse large B-cell lymphoma (DLBCL). Twenty-nine percent of activated B-cell-type DLBCL (ABC-DLBCL), which is characterized by constitutive activation of the NF-κB pathway, carry the p.L265P mutation. In addition, ABC-DLBCL frequently displays focal copy number gains affecting BCL2 Here, we generated a novel mouse model in which Cre-mediated recombination, specifically in B cells, leads to the conditional expression of Myd88(p.L252P) (the orthologous position of the human MYD88(p.L265P) mutation) from the endogenous locus. These mice develop a lymphoproliferative disease and occasional transformation into clonal lymphomas. The clonal disease displays the morphologic and immunophenotypical characteristics of ABC-DLBCL. Lymphomagenesis can be accelerated by crossing in a further novel allele, which mediates conditional overexpression of BCL2 Cross-validation experiments in human DLBCL samples revealed that both MYD88 and CD79B mutations are substantially enriched in ABC-DLBCL compared with germinal center B-cell DLBCL. Furthermore, analyses of human DLBCL genome sequencing data confirmed that BCL2 amplifications frequently co-occurred with MYD88 mutations, further validating our approach. Finally, in silico experiments revealed that MYD88-mutant ABC-DLBCL cells in particular display an actionable addiction to BCL2. Altogether, we generated a novel autochthonous mouse model of ABC-DLBCL that could be used as a preclinical platform for the development and validation of novel therapeutic approaches for the treatment of ABC-DLBCL.

Human NACHT, LRR, and PYD domain-containing protein 3 (NLRP3) inflammasome activity is regulated by and potentially targetable through Bruton tyrosine kinase.

BACKGROUND: The Nod-like receptor NACHT, LRR, and PYD domain-containing protein 3 (NLRP3) and Bruton tyrosine kinase (BTK) are protagonists in innate and adaptive immunity, respectively. NLRP3 senses exogenous and endogenous insults, leading to inflammasome activation, which occurs spontaneously in patients with Muckle-Wells syndrome; BTK mutations cause the genetic immunodeficiency X-linked agammaglobulinemia (XLA). However, to date, few proteins that regulate NLRP3 inflammasome activity in human primary immune cells have been identified, and clinically promising pharmacologic targeting strategies remain elusive. OBJECTIVE: We sought to identify novel regulators of the NLRP3 inflammasome in human cells with a view to exploring interference with inflammasome activity at the level of such regulators. METHODS: After proteome-wide phosphoproteomics, the identified novel regulator BTK was studied in human and murine cells by using pharmacologic and genetic BTK ablation. RESULTS: Here we show that BTK is a critical regulator of NLRP3 inflammasome activation: pharmacologic (using the US Food and Drug Administration-approved inhibitor ibrutinib) and genetic (in patients with XLA and Btk knockout mice) BTK ablation in primary immune cells led to reduced IL-1ß processing and secretion in response to nigericin and the Staphylococcus aureus toxin leukocidin AB (LukAB). BTK affected apoptosis-associated speck-like protein containing a CARD (ASC) speck formation and caspase-1 cleavage and interacted with NLRP3 and ASC. S aureus infection control in vivo and IL-1ß release from cells of patients with Muckle-Wells syndrome were impaired by ibrutinib. Notably, IL-1ß processing and release from immune cells isolated from patients with cancer receiving ibrutinib therapy were reduced. CONCLUSION: Our data suggest that XLA might result in part from genetic inflammasome deficiency and that NLRP3 inflammasome-linked inflammation could potentially be targeted pharmacologically through BTK.

A frequent hypofunctional IRAK2 variant is associated with reduced spontaneous hepatitis C virus clearance.

UNLABELLED: Patients carrying very rare loss-of-function mutations in interleukin-1 receptor-associated kinase 4 (IRAK4), a critical signaling mediator in Toll-like receptor signaling, are severely immunodeficient, highlighting the paramount role of IRAK kinases in innate immunity. We discovered a comparatively frequent coding variant of the enigmatic human IRAK2, L392V (rs3844283), which is found homozygously in ∼15% of Caucasians, to be associated with a reduced ability to induce interferon-alpha in primary human plasmacytoid dendritic cells in response to hepatitis C virus (HCV). Cytokine production in response to purified Toll-like receptor agonists was also impaired. Additionally, rs3844283 was epidemiologically associated with a chronic course of HCV infection in two independent HCV cohorts and emerged as an independent predictor of chronic HCV disease. Mechanistically, IRAK2 L392V showed intact binding to, but impaired ubiquitination of, tumor necrosis factor receptor-associated factor 6, a vital step in signal transduction. CONCLUSION: Our study highlights IRAK2 and its genetic variants as critical factors and potentially novel biomarkers for human antiviral innate immunity.

Activation of lymphoma-associated MyD88 mutations via allostery-induced TIR-domain oligomerization.

Myeloid differentiation 88 (MyD88) is the key signaling adapter of Toll-like and interleukin-1 receptors. Recurrent lymphoma-associated mutations, particularly Leu265Pro (L265P), within the MyD88 Toll/interleukin-1 receptor (TIR) domain sustain lymphoma cell survival due to constitutive nuclear factor κB signaling. We found that mutated TIR domains displayed an intrinsic propensity for augmented oligomerization and spontaneous formation of cytosolic Myddosome aggregates in lymphoma cell lines, mimicking the effect of dimerized TIR domains. Blocking of MyD88 oligomerization induced apoptosis. The L265P TIR domain can recruit the endogenous wild-type MyD88 for oligomer formation and hyperactivity. Molecular dynamics simulations and analysis of additional mutations suggest that constitutive activity is caused by allosteric oligomerization.

Innate Responses to the Former COVID-19 Vaccine Candidate CVnCoV and Their Relation to Reactogenicity and Adaptive Immunogenicity.

Vaccines are highly effective at preventing severe coronavirus disease (COVID-19). With mRNA vaccines, further research is needed to understand the association between immunogenicity and reactogenicity, which is defined as the physical manifestation of an inflammatory response to a vaccination. This study analyzed the immune response and reactogenicity in humans, post immunization, to the former SARS-CoV-2 mRNA investigational vaccine CVnCoV (CV-NCOV-001 and CV-NCOV-002 clinical trials). Immunogenicity was investigated using whole-blood RNA sequencing, serum cytokine levels, and SARS-CoV-2-specific antibodies. The T cell responses in peripheral blood were assessed using intracellular cytokine staining (ICS) and high-dimensional profiling in conjunction with SARS-CoV-2 antigen-specificity testing via mass cytometry. Reactogenicity was graded after participants' first and second doses of CVnCoV using vaccine-related solicited adverse events (AEs). Finally, a Spearman correlation was performed between reactogenicity, humoral immunity, and serum cytokine levels to assess the relationship between reactogenicity and immunogenicity post CVnCoV vaccination. Our findings showed that the gene sets related to innate and inflammatory immune responses were upregulated one day post CVnCoV vaccination, while the gene sets related to adaptive immunity were upregulated predominantly one week after the second dose. The serum levels of IFNα, IFNγ, IP-10, CXCL11, IL-10, and MCP-1 increased transiently, peaking one day post vaccination. CD4+ T cells were induced in all vaccinated participants and low frequencies of CD8+ T cells were detected by ex vivo ICS. Using mass cytometry, SARS-CoV-2 spike-specific CD8+ T cells were induced and were characterized as having an activated effector memory phenotype. Overall, the results demonstrated a positive correlation between vaccine-induced systemic cytokines, reactogenicity, and adaptive immunity, highlighting the importance of the balance between the induction of innate immunity to achieve vaccine efficacy and ensuring low reactogenicity.

Corrigendum to "Safety and immunogenicity of mRNA-LNP COVID-19 vaccine CVnCoV in Latin American adults: A phase 2 randomized study" [Vaccine: X 11 (2022) 100189].

[This corrects the article DOI: 10.1016/j.jvacx.2022.100189.].

A Third Dose of the COVID-19 Vaccine, CVnCoV, Increased the Neutralizing Activity against the SARS-CoV-2 Wild-Type and Delta Variant.

A third dose of CVnCoV, a former candidate mRNA vaccine against SARS-CoV-2, was previously shown to boost neutralizing antibody responses against SARS-CoV-2 wild-type in adults aged 18−60 and >60 years in a phase 2a clinical study. In the present study, we report the neutralizing antibody responses to a wild-type and a variant of concern, Delta, after a third dose of the vaccine on day (D)57 and D180. Neutralization activity was assessed using a microneutralization assay. Comparable levels of neutralizing antibodies against the wild-type and Delta were induced. These were higher than those observed after the first two doses, irrespective of age or pre-SARS-CoV-2-exposure status, indicating that the first two doses induced immune memory. Four weeks after the third dose on D180, the neutralizing titers for wild-type and Delta were two-fold higher in younger participants than in older participants; seroconversion rates were 100% for wild-type and Delta in the younger group and for Delta in the older group. A third CVnCoV dose induced similar levels of neutralizing responses against wild-type virus and the Delta variant in both naïve and pre-exposed participants, aligning with current knowledge from licensed COVID-19 vaccines that a third dose is beneficial against SARS-CoV-2 variants.

Safety and immunogenicity of mRNA-LNP COVID-19 vaccine CVnCoV in Latin American adults: A phase 2 randomized study.

Background: The COVID-19 vaccine candidate CVnCoV comprises sequence-optimized mRNA encoding SARS-CoV-2 S-protein encapsulated in lipid nanoparticles. In this phase 2a study, we assessed reactogenicity and immunogenicity of two or three doses in younger and older adults. Methods: Younger (18-60 years) and older (>60 years) adults were enrolled in two sites in Panama and Peru to receive either 6 or 12 µg doses of CVnCoV or licensed control vaccines 28 days apart; subsets received a 12 µg booster dose on Day 57 or Day 180. Solicited adverse events (AE) were reported for 7 days and unsolicited AEs for 4 weeks after each vaccination, and serious AEs (SAE) throughout the study. Humoral immunogenicity was measured as neutralizing and receptor binding domain (RBD) IgG antibodies and cellular immunogenicity was assessed as CD4+/CD8 + T cell responses. Results: A total of 668 participants were vaccinated (332 aged 18-60 years and 336 aged > 60 years) including 75 who received homologous booster doses. Vaccination was well tolerated with no vaccine-related SAEs. Solicited and unsolicited AEs were mainly mild to moderate and resolved spontaneously. Both age groups demonstrated robust immune responses as neutralizing antibodies or RBD-binding IgG, after two doses, with lower titers in the older age group than the younger adults. Neither group achieved levels observed in human convalescent sera (HCS), but did equal or surpass HCS levels following homologous booster doses. Following CVnCoV vaccination, robust SARS-CoV-2 S-protein-specific CD4 + T-cell responses were observed in both age groups with CD8 + T-cell responses in some individuals, consistent with observations in convalescing COVID-19 patients after natural infection. Conclusions: We confirmed that two 12 µg doses of CVnCoV had an acceptable safety profile, and induced robust immune responses. Marked humoral immune responses to homologous boosters suggest two doses had induced immune memory.

Absence of Non-Canonical, Inhibitory MYD88 Splice Variants in B Cell Lymphomas Correlates With Sustained NF-κB Signaling.

Gain-of-function mutations of the TLR adaptor and oncoprotein MyD88 drive B cell lymphomagenesis via sustained NF-κB activation. In myeloid cells, both short and sustained TLR activation and NF-κB activation lead to the induction of inhibitory MYD88 splice variants that restrain prolonged NF-κB activation. We therefore sought to investigate whether such a negative feedback loop exists in B cells. Analyzing MYD88 splice variants in normal B cells and different primary B cell malignancies, we observed that MYD88 splice variants in transformed B cells are dominated by the canonical, strongly NF-κB-activating isoform of MYD88 and contain at least three novel, so far uncharacterized signaling-competent splice isoforms. Sustained TLR stimulation in B cells unexpectedly reinforces splicing of NF-κB-promoting, canonical isoforms rather than the 'MyD88s', a negative regulatory isoform reported to be typically induced by TLRs in myeloid cells. This suggests that an essential negative feedback loop restricting TLR signaling in myeloid cells at the level of alternative splicing, is missing in B cells when they undergo proliferation, rendering B cells vulnerable to sustained NF-κB activation and eventual lymphomagenesis. Our results uncover MYD88 alternative splicing as an unappreciated promoter of B cell lymphomagenesis and provide a rationale why oncogenic MYD88 mutations are exclusively found in B cells.

Safety and immunogenicity of an mRNA-lipid nanoparticle vaccine candidate against SARS-CoV-2 : A phase 1 randomized clinical trial.

BACKGROUND: We used the RNActive® technology platform (CureVac N.V., Tübingen, Germany) to prepare CVnCoV, a COVID-19 vaccine containing sequence-optimized mRNA coding for a stabilized form of SARS-CoV­2 spike (S) protein encapsulated in lipid nanoparticles (LNP). METHODS: This is an interim analysis of a dosage escalation phase 1 study in healthy 18-60-year-old volunteers in Hannover, Munich and Tübingen, Germany, and Ghent, Belgium. After giving 2 intramuscular doses of CVnCoV or placebo 28 days apart we assessed solicited local and systemic adverse events (AE) for 7 days and unsolicited AEs for 28 days after each vaccination. Immunogenicity was measured as enzyme-linked immunosorbent assay (ELISA) IgG antibodies to SARS-CoV­2 S­protein and receptor binding domain (RBD), and SARS-CoV­2 neutralizing titers (MN50). RESULTS: In 245 volunteers who received 2 CVnCoV vaccinations (2 µg, n = 47, 4 µg, n = 48, 6 µg, n = 46, 8 µg, n = 44, 12 µg, n = 28) or placebo (n = 32) there were no vaccine-related serious AEs. Dosage-dependent increases in frequency and severity of solicited systemic AEs, and to a lesser extent local AEs, were mainly mild or moderate and transient in duration. Dosage-dependent increases in IgG antibodies to S­protein and RBD and MN50 were evident in all groups 2 weeks after the second dose when 100% (23/23) seroconverted to S­protein or RBD, and 83% (19/23) seroconverted for MN50 in the 12 µg group. Responses to 12 µg were comparable to those observed in convalescent sera from known COVID-19 patients. CONCLUSION: In this study 2 CVnCoV doses were safe, with acceptable reactogenicity and 12 µg dosages elicited levels of immune responses that overlapped those observed in convalescent sera.

BTK operates a phospho-tyrosine switch to regulate NLRP3 inflammasome activity.

Activity of the NLRP3 inflammasome, a critical mediator of inflammation, is controlled by accessory proteins, posttranslational modifications, cellular localization, and oligomerization. How these factors relate is unclear. We show that a well-established drug target, Bruton's tyrosine kinase (BTK), affects several levels of NLRP3 regulation. BTK directly interacts with NLRP3 in immune cells and phosphorylates four conserved tyrosine residues upon inflammasome activation, in vitro and in vivo. Furthermore, BTK promotes NLRP3 relocalization, oligomerization, ASC polymerization, and full inflammasome assembly, probably by charge neutralization, upon modification of a polybasic linker known to direct NLRP3 Golgi association and inflammasome nucleation. As NLRP3 tyrosine modification by BTK also positively regulates IL-1ß release, we propose BTK as a multifunctional positive regulator of NLRP3 regulation and BTK phosphorylation of NLRP3 as a novel and therapeutically tractable step in the control of inflammation.

Putative link between Polo-like kinases (PLKs) and Toll-like receptor (TLR) signaling in transformed and primary human immune cells.

Toll-like receptors (TLRs) are important sentinels of bacterial and viral infection and thus fulfil a critical sensory role in innate immunity. Polo-like kinases (PLKs), a five membered family of Ser/Thr protein kinases, have long been studied for their role in mitosis and thus represent attractive therapeutic targets in cancer therapy. Recently, PLKs were implicated in TLR signaling in mice but the role of PLKs in TLR signaling in untransformed primary immune cells has not been addressed, even though PLK inhibitors are in clinical trials. We here identified several phospho-serine and phospho-threonine residues in the known TLR pathway kinases, Interleukin-1 receptor-associated kinase (IRAK) 2 and IRAK4. These sites lie in canonical polo-box motifs (PBM), sequence motifs known to direct recruitment of PLKs to client proteins. Interestingly, PLK1 was phosphorylated and PLK 2 and 3 mRNA induced upon TLR stimulation in primary immune cells, respectively. In whole blood, PLK inhibition disparately affected TLR mediated cytokine responses in a donor- and inhibitor-dependent fashion. Collectively, PLKs may thus potentially interface with TLR signaling in humans. We propose that temporary PLK inhibitor-mediated blockade of TLR-signaling in certain patients receiving such inhibitors during cancer treatment may cause adverse effects such as an increased risk of infections due to a then compromised ability of the TLR recognition system to sense and initiate cytokine responses to invading microbes.

Examining Myddosome Formation by Luminescence-Based Mammalian Interactome Mapping (LUMIER).

Recent structural, biochemical, and functional studies have led to the notion that many of the post-receptor signaling complexes in innate immunity have a multimeric, multi-protein architecture whose hierarchical assembly is vital for function. The Myddosome is a post-receptor complex in the cytoplasmic signaling of Toll-like receptors (TLR) and the Interleukin-1 receptor (IL-1R), involving the proteins MyD88, IL-1R-associated kinase 4 (IRAK4), and IRAK2. Its importance is strikingly illustrated by the fact that rare germline mutations in MYD88 causing high susceptibility to infections are characterized by failure to assemble Myddosomes; conversely, gain-of-function MYD88 mutations leading to oncogenic hyperactivation of NF-κB show increased Myddosome formation. Reliable methods to probe Myddosome formation experimentally are therefore vital to further study the properties of this important post-receptor complex and its role in innate immunity, such as its regulation by posttranslational modification. Compared to structural and biochemical analyses, luminescence-based mammalian interactome mapping (LUMIER) is a straightforward, automatable, quantifiable, and versatile technique to study protein-protein interactions in a physiologically relevant context. We adapted LUMIER for Myddosome analysis and provide here a basic background of this technique, suitable experimental protocols, and its potential for medium-throughput screening. The principles presented herein can be adapted to other signaling pathways.

HLA class I-restricted MYD88 L265P-derived peptides as specific targets for lymphoma immunotherapy.

Genome sequencing has uncovered an array of recurring somatic mutations in different non-Hodgkin lymphoma (NHL) subtypes. If affecting protein-coding regions, such mutations may yield mutation-derived peptides that may be presented by HLA class I proteins and recognized by cytotoxic T cells. A recurring somatic and oncogenic driver mutation of the Toll-like receptor adaptor protein MYD88, Leu265Pro (L265P) was identified in up to 90% of different NHL subtype patients. We therefore screened the potential of MYD88L265P-derived peptides to elicit cytotoxic T cell responses as tumor-specific neoantigens. Based on in silico predictions, we identified potential MYD88L265P-containing HLA ligands for several HLA class I restrictions. A set of HLA class I MYD88L265P-derived ligands elicited specific cytotoxic T cell responses for HLA-B*07 and -B*15. These data highlight the potential of MYD88L265P mutation-specific peptide-based immunotherapy as a novel personalized treatment approach for patients with MYD88L265P+ NHLs that may complement pharmacological approaches targeting oncogenic MyD88 L265P signaling.