The bacterial lysate OM-85 engages Toll-like receptors 2 and 4 triggering an immunomodulatory gene signature in human myeloid cells.

OM-85 is a bacterial lysate used in clinical practice to reduce duration and frequency of recurrent respiratory tract infections. Whereas knowledge of its regulatory effects in vivo has substantially advanced, the mechanisms of OM-85 sensing remain inadequately addressed. Here, we show that the immune response to OM-85 in the mouse is largely mediated by myeloid immune cells through Toll-like receptor (TLR) 4 in vitro and in vivo. Instead, in human immune cells, TLR2 and TLR4 orchestrate the response to OM-85, which binds to both receptors as shown by surface plasmon resonance assay. Ribonucleic acid-sequencing analyses of human monocyte-derived dendritic cells reveal that OM-85 triggers a pro-inflammatory signature and a unique gene set, which is not induced by canonical agonists of TLR2 or TLR4 and comprises tolerogenic genes. A largely overlapping TLR2/4-dependent gene signature was observed in individual subsets of primary human airway myeloid cells, highlighting the robust effects of OM-85. Collectively, our results suggest caution should be taken when relating murine studies on bacterial lysates to humans. Furthermore, our data shed light on how a standardized bacterial lysate shapes the response through TLR2 and TLR4, which are crucial for immune response, trained immunity, and tolerance.

SARS-CoV-2 infection and cognition in community-dwelling and nursing home residents in southern Switzerland.

Background: COVID-19 patients can report 'brain fog' and may exhibit cognitive symptoms for months after recovery (Cognitive COVID). However, evidence on whether and the extent to which SARS-CoV-2 infection impacts cognition irrespective of COVID-19 course and severity is limited to clinical samples and mainly comes from prognostic studies. We aimed to explore the association between serologically confirmed SARS-CoV-2 infection and cognitive functioning in community-based and institutionalized older adults, irrespective of COVID-19 symptoms. Methods: We conducted a case-control study nested into two cohorts in Southern Switzerland. Eligible subjects were Italian speaking older adults, without a previous diagnosis of dementia, who underwent serological testing for anti-SARS-CoV-2 antibodies between November 2020 and July 2021. We manually selected age-, sex- and education-matched cases (i.e., individuals with a serologically confirmed SARS-CoV-2 infection), with seronegative controls, and we conducted in-person neuropsychological assessments using validated, highly sensitive cognitive tests. Results: We completed 38 neuropsychological assessments in a mostly female sample of older adults (Mean age: 83.13 ± 8.95; 86.8% women). 17 were community dwelling individuals while 21 lived in a nursing home. As expected, socio-demographic characteristics of age, gender and educational level were similarly distributed between cases (n = 14) and controls (n = 24). In linear regression models, cases had significantly lower scores in cognitive tasks of memory (ß = -0.367, p = 0.023), attention (ß = 0.428, p = 0.008) and executive functions (ß = 0.326, p = 0.046). We found no significant difference in tests of language and spatial-temporal orientation (all p values > 0.05). Conclusions: SARS-CoV-2 infection was associated with cognitive impairment in memory, attention, and executive functions in older adults. Our findings are consistent with mechanistic evidence of the neurotropism of the virus and provide empirical support for the "Cognitive COVID" construct also in non-clinical samples. With nearly 800 million COVID-19 cases (in April 2023), and many more infections worldwide, the clinical and public health implications of Cognitive COVID due to SARS-CoV-2 infection may be massive and warrant further epidemiological investigations.

Human MCTS1-dependent translation of JAK2 is essential for IFN-γ immunity to mycobacteria.

Human inherited disorders of interferon-gamma (IFN-γ) immunity underlie severe mycobacterial diseases. We report X-linked recessive MCTS1 deficiency in men with mycobacterial disease from kindreds of different ancestries (from China, Finland, Iran, and Saudi Arabia). Complete deficiency of this translation re-initiation factor impairs the translation of a subset of proteins, including the kinase JAK2 in all cell types tested, including T lymphocytes and phagocytes. JAK2 expression is sufficiently low to impair cellular responses to interleukin-23 (IL-23) and partially IL-12, but not other JAK2-dependent cytokines. Defective responses to IL-23 preferentially impair the production of IFN-γ by innate-like adaptive mucosal-associated invariant T cells (MAIT) and γδ T lymphocytes upon mycobacterial challenge. Surprisingly, the lack of MCTS1-dependent translation re-initiation and ribosome recycling seems to be otherwise physiologically redundant in these patients. These findings suggest that X-linked recessive human MCTS1 deficiency underlies isolated mycobacterial disease by impairing JAK2 translation in innate-like adaptive T lymphocytes, thereby impairing the IL-23-dependent induction of IFN-γ.

Regulatory circuits of mitophagy restrict distinct modes of cell death during memory CD8+ T cell formation.

Mitophagy, a central process guarding mitochondrial quality, is commonly impaired in human diseases such as Parkinson's disease, but its impact in adaptive immunity remains unclear. The differentiation and survival of memory CD8+ T cells rely on oxidative metabolism, a process that requires robust mitochondrial quality control. Here, we found that Parkinson's disease patients have a reduced frequency of CD8+ memory T cells compared with healthy donors and failed to form memory T cells upon vaccination against COVID-19, highlighting the importance of mitochondrial quality control for memory CD8+ T cell formation. We further uncovered that regulators of mitophagy, including Parkin and NIX, were up-regulated in response to interleukin-15 (IL-15) for supporting memory T cell formation. Mechanistically, Parkin suppressed VDAC1-dependent apoptosis in memory T cells. In contrast, NIX expression in T cells counteracted ferroptosis by preventing metabolic dysfunction resulting from impaired mitophagy. Together, our results indicate that the mitophagy machinery orchestrates survival and metabolic dynamics required for memory T cell formation, as well as highlight a deficit in T cell-mediated antiviral responses in Parkinson's disease patients.

The interplay between previous infection and mental health condition on antibody response to COVID-19 mRNA vaccination.

Increasing evidence has been pointing towards the existence of a bi-directional interplay between mental health condition and immunity. Data collected during the COVID-19 outbreak suggest that depressive symptoms may impact the production of antibodies against SARS-CoV-2, while a previous infection could affect the immune response and cause neuropsychological disturbances. A prospective observational study was designed to investigate the association between mental health conditions and immune response over time. We analyzed the mental health at baseline and the antibodies before and after immunization with the COVID-19 mRNA vaccine in a cohort of healthcare professionals in southern Switzerland. One-hundred and six subjects were enrolled. Anxiety, distress and depression correlated to each other. There were no correlations between the mentioned variables and the vaccine induced IgG antibodies against the receptor binding domain (RBD) of the spike protein. For those who had a previous COVID-19 infection, the antibodies increased according to the grade of depression. For those who did not, the anti-RBD IgG levels remained similar when comparing presence or absence of depression symptoms. Our results show that previous SARS-CoV-2 natural infection in subjects with mental health conditions enhances the immune response to COVID-19 mRNA vaccination. The correlation between immune response to COVID-19 vaccination, a previous exposure to the virus, and symptoms of mood disorders, makes it necessary to explore the direction of the causality between immune response and depressive symptoms.

Machine learning analysis of humoral and cellular responses to SARS-CoV-2 infection in young adults.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induces B and T cell responses, contributing to virus neutralization. In a cohort of 2,911 young adults, we identified 65 individuals who had an asymptomatic or mildly symptomatic SARS-CoV-2 infection and characterized their humoral and T cell responses to the Spike (S), Nucleocapsid (N) and Membrane (M) proteins. We found that previous infection induced CD4 T cells that vigorously responded to pools of peptides derived from the S and N proteins. By using statistical and machine learning models, we observed that the T cell response highly correlated with a compound titer of antibodies against the Receptor Binding Domain (RBD), S and N. However, while serum antibodies decayed over time, the cellular phenotype of these individuals remained stable over four months. Our computational analysis demonstrates that in young adults, asymptomatic and paucisymptomatic SARS-CoV-2 infections can induce robust and long-lasting CD4 T cell responses that exhibit slower decays than antibody titers. These observations imply that next-generation COVID-19 vaccines should be designed to induce stronger cellular responses to sustain the generation of potent neutralizing antibodies.

Antibodies Produced by CLL Phenotype B Cells in Patients With Myasthenia Gravis Are Not Directed Against Neuromuscular Endplates.

BACKGROUND AND OBJECTIVES: Myasthenia gravis (MG) can in rare cases be an autoimmune phenomenon associated with hematologic malignancies such as chronic lymphocytic leukemia (CLL). It is unclear whether in patients with MG and CLL, the leukemic B cells are the ones directly driving the autoimmune response against neuromuscular endplates. METHODS: We identified patients with acetylcholine receptor antibody-positive (AChR+) MG and CLL or monoclonal B-cell lymphocytosis (MBL), a precursor to CLL, and described their clinical features, including treatment responses. We generated recombinant monoclonal antibodies (mAbs) corresponding to the B-cell receptors of the CLL phenotype B cells and screened them for autoantigen binding. RESULTS: A computational immune cell screen revealed a subgroup of 5/38 patients with MG and 0/21 healthy controls who displayed a CLL-like B-cell phenotype. In follow-up hematologic flow cytometry, 2 of these 5 patients were diagnosed with an MBL. An additional patient with AChR+ MG as a complication of manifest CLL presented at our neuromuscular clinic and was successfully treated with the anti-CD20 therapy obinutuzumab plus chlorambucil. We investigated the specificities of expanding CLL-like B-cell clones to assess a direct causal link between the 2 diseases. However, we observed no reactivity of the clones against the AChR, antigens at the neuromuscular junction, or other common autoantigens. DISCUSSION: Our study suggests that AChR autoantibodies are produced by nonmalignant, polyclonal B cells The new anti-CD20 treatment obinutuzumab might be considered in effectively treating AChR+ MG. CLASSIFICATION OF EVIDENCE: This is a single case study and provides Class IV evidence that obinutuzumab is safe to use in patients with MG.

Maturation of SARS-CoV-2 Spike-specific memory B cells drives resilience to viral escape.

Memory B cells (MBCs) generate rapid antibody responses upon secondary encounter with a pathogen. Here, we investigated the kinetics, avidity, and cross-reactivity of serum antibodies and MBCs in 155 SARS-CoV-2 infected and vaccinated individuals over a 16-month time frame. SARS-CoV-2-specific MBCs and serum antibodies reached steady-state titers with comparable kinetics in infected and vaccinated individuals. Whereas MBCs of infected individuals targeted both prefusion and postfusion Spike (S), most vaccine-elicited MBCs were specific for prefusion S, consistent with the use of prefusion-stabilized S in mRNA vaccines. Furthermore, a large fraction of MBCs recognizing postfusion S cross-reacted with human betacoronaviruses. The avidity of MBC-derived and serum antibodies increased over time resulting in enhanced resilience to viral escape by SARS-CoV-2 variants, including Omicron BA.1 and BA.2 sublineages, albeit only partially for BA.4 and BA.5 sublineages. Overall, the maturation of high-affinity and broadly reactive MBCs provides the basis for effective recall responses to future SARS-CoV-2 variants.

Longitudinal Postvaccine SARS-CoV-2 Immunoglobulin G Titers, Memory B-Cell Responses, and Risk of COVID-19 in Multiple Sclerosis Over 1 Year.

BACKGROUND AND OBJECTIVES: Some disease-modifying treatments impair response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines in multiple sclerosis (MS), potentially increasing the risk of breakthrough infections. We aimed to investigate longitudinal SARS-CoV-2 antibody dynamics and memory B cells after 2 and 3 messenger RNA (mRNA) vaccine doses and their association with the risk of COVID-19 in patients with MS on different treatments over 1 year. METHODS: Prospective observational cohort study in patients with MS undergoing SARS-CoV-2 mRNA vaccinations. Antispike (anti-S) immunoglobulin G (IgG) titers were measured by chemiluminescence microparticle immunoassay. Frequencies of spike-specific memory B cells were measured on polyclonal stimulation of peripheral blood mononuclear cells and screening of secreted antibodies by ELISA. RESULTS: We recruited 120 patients with MS (58 on anti-CD20 antibodies, 9 on sphingosine 1-phosphate (S1P) receptor modulators, 15 on cladribine, 24 on teriflunomide (TFL), and 14 untreated) and collected 392 samples up to 10.8 months after 2 vaccine doses. When compared with untreated patients, anti-CD20 antibodies (ß = -2.07, p < 0.001) and S1P modulators (ß = -2.02, p < 0.001) were associated with lower anti-S IgG, while TFL and cladribine were not. Anti-S IgG decreased with months since vaccine (ß = -0.14, p < 0.001), independently of treatments. Within anti-CD20 patients, anti-S IgG remained higher in those with greater baseline B-cell counts and were not influenced by postvaccine anti-CD20 infusions. Anti-S IgG increase after a 3rd vaccine was mild on anti-CD20 and S1P modulators. Spike-specific memory B-cell responses were weaker on S1P modulators and anti-CD20 than on TFL and influenced by postvaccine anti-CD20 infusions. The frequency of breakthrough infections was comparable between DMTs, but the risk of COVID-19 was predicted by the last measured anti-S IgG titer before infection (OR = 0.56, 95% CI = 0.37-0.86, p = 0.008). DISCUSSION: Postvaccine anti-S IgG titers decrease over time regardless of MS treatment and are associated with breakthrough COVID-19. Both humoral and specific memory B-cell responses are diminished on S1P modulators. Within anti-CD20-treated patients, B-cell count at first vaccine determines anti-S IgG production, whereas postvaccine anti-CD20 infusions negatively affect spike-specific memory B cells.

Clonal composition and persistence of antigen-specific circulating T follicular helper cells.

T follicular helper (TFH ) cells play an essential role in promoting B cell responses and antibody affinity maturation in germinal centers (GC). A subset of memory CD4+ T cells expressing the chemokine receptor CXCR5 has been described in human blood as phenotypically and clonally related to GC TFH cells. However, the antigen specificity and relationship of these circulating TFH (cTFH ) cells with other memory CD4+ T cells remain poorly defined. Combining antigenic stimulation and T cell receptor (TCR) Vß sequencing, we found T cells specific to tetanus toxoid (TT), influenza vaccine (Flu), or Candida albicans (C.alb) in both cTFH and non-cTFH subsets, although with different frequencies and effector functions. Interestingly, cTFH and non-cTFH cells specific for C.alb or TT had a largely overlapping TCR Vß repertoire while the repertoire of Flu-specific cTFH and non-cTFH cells was distinct. Furthermore, Flu-specific but not C.alb-specific PD-1+ cTFH cells had a "GC TFH -like" phenotype, with overexpression of IL21, CXCL13, and BCL6. Longitudinal analysis of serial blood donations showed that Flu-specific cTFH and non-cTFH cells persisted as stable repertoires for years. Collectively, our study provides insights on the relationship of cTFH with non-cTFH cells and on the heterogeneity and persistence of antigen-specific human cTFH cells.

SARS-CoV-2 spike conformation determines plasma neutralizing activity elicited by a wide panel of human vaccines.

Numerous safe and effective coronavirus disease 2019 vaccines have been developed worldwide that use various delivery technologies and engineering strategies. We show here that vaccines containing prefusion-stabilizing S mutations elicit antibody responses in humans with enhanced recognition of S and the S1 subunit relative to postfusion S as compared with vaccines lacking these mutations or natural infection. Prefusion S and S1 antibody binding titers positively and equivalently correlated with neutralizing activity, and depletion of S1-directed antibodies completely abrogated plasma neutralizing activity. We show that neutralizing activity is almost entirely directed to the S1 subunit and that variant cross-neutralization is mediated solely by receptor binding domain-specific antibodies. Our data provide a quantitative framework for guiding future S engineering efforts to develop vaccines with higher resilience to the emergence of variants than current technologies.

Maturation of SARS-CoV-2 Spike-specific memory B cells drives resilience to viral escape.

Memory B cells (MBCs) generate rapid antibody responses upon secondary encounter with a pathogen. Here, we investigated the kinetics, avidity and cross-reactivity of serum antibodies and MBCs in 155 SARS-CoV-2 infected and vaccinated individuals over a 16-month timeframe. SARS-CoV-2-specific MBCs and serum antibodies reached steady-state titers with comparable kinetics in infected and vaccinated individuals. Whereas MBCs of infected individuals targeted both pre- and postfusion Spike (S), most vaccine-elicited MBCs were specific for prefusion S, consistent with the use of prefusion-stabilized S in mRNA vaccines. Furthermore, a large fraction of MBCs recognizing postfusion S cross-reacted with human betacoronaviruses. The avidity of MBC-derived and serum antibodies increased over time resulting in enhanced resilience to viral escape by SARS-CoV-2 variants, including Omicron BA.1 and BA.2 sub-lineages, albeit only partially for BA.4 and BA.5 sublineages. Overall, the maturation of high-affinity and broadly-reactive MBCs provides the basis for effective recall responses to future SARS-CoV-2 variants.

PDIA3 epitope-driven immune autoreactivity contributes to hepatic damage in type 2 diabetes.

A diet rich in saturated fat and carbohydrates causes low-grade chronic inflammation in several organs, including the liver, ultimately driving nonalcoholic steatohepatitis. In this setting, environment-driven lipotoxicity and glucotoxicity induce liver damage, which promotes dendritic cell activation and generates a major histocompatibility complex class II (MHC-II) immunopeptidome enriched with peptides derived from proteins involved in cellular metabolism, oxidative phosphorylation, and the stress responses. Here, we demonstrated that lipotoxicity and glucotoxicity, as driven by a high-fat and high-fructose (HFHF) diet, promoted MHC-II presentation of nested T and B cell epitopes from protein disulfide isomerase family A member 3 (PDIA3), which is involved in immunogenic cell death. Increased MHC-II presentation of PDIA3 peptides was associated with antigen-specific proliferation of hepatic CD4+ immune infiltrates and isotype switch of anti-PDIA3 antibodies from IgM to IgG3, indicative of cellular and humoral PDIA3 autoreactivity. Passive transfer of PDIA3-specific T cells or PDIA3-specific antibodies also exacerbated hepatocyte death, as determined by increased hepatic transaminases detected in the sera of mice subjected to an HFHF but not control diet. Increased humoral responses to PDIA3 were also observed in patients with chronic inflammatory liver conditions, including autoimmune hepatitis, primary biliary cholangitis, and type 2 diabetes. Together, our data indicated that metabolic insults caused by an HFHF diet elicited liver damage and promoted pathogenic immune autoreactivity driven by T and B cell PDIA3 epitopes.

ACE2-binding exposes the SARS-CoV-2 fusion peptide to broadly neutralizing coronavirus antibodies.

The coronavirus spike glycoprotein attaches to host receptors and mediates viral fusion. Using a broad screening approach, we isolated seven monoclonal antibodies (mAbs) that bind to all human-infecting coronavirus spike proteins from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immune donors. These mAbs recognize the fusion peptide and acquire affinity and breadth through somatic mutations. Despite targeting a conserved motif, only some mAbs show broad neutralizing activity in vitro against alpha- and betacoronaviruses, including animal coronaviruses WIV-1 and PDF-2180. Two selected mAbs also neutralize Omicron BA.1 and BA.2 authentic viruses and reduce viral burden and pathology in vivo. Structural and functional analyses showed that the fusion peptide-specific mAbs bound with different modalities to a cryptic epitope hidden in prefusion stabilized spike, which became exposed upon binding of angiotensin-converting enzyme 2 (ACE2) or ACE2-mimicking mAbs.

Clonal structure, stability and dynamics of human memory B cells and circulating plasmablasts.

Memory B cells persist for a lifetime and rapidly differentiate into antibody-producing plasmablasts and plasma cells upon antigen re-encounter. The clonal relationship and evolution of memory B cells and circulating plasmablasts is not well understood. Using single-cell sequencing combined with isolation of specific antibodies, we found that in two healthy donors, the memory B cell repertoire was dominated by large IgM, IgA and IgG2 clonal families, whereas IgG1 families, including those specific for recall antigens, were of small size. Analysis of multiyear samples demonstrated stability of memory B cell clonal families and revealed that a large fraction of recently generated plasmablasts was derived from long-term memory B cell families and was found recurrently. Collectively, this study provides a systematic description of the structure, stability and dynamics of the human memory B cell pool and suggests that memory B cells may be active at any time point in the generation of plasmablasts.

Antibodies to a Citrullinated Porphyromonas gingivalis Epitope Are Increased in Early Rheumatoid Arthritis, and Can Be Produced by Gingival Tissue B Cells: Implications for a Bacterial Origin in RA Etiology.

Based on the epidemiological link between periodontitis and rheumatoid arthritis (RA), and the unique feature of the periodontal bacterium Porphyromonas gingivalis to citrullinate proteins, it has been suggested that production of anti-citrullinated protein antibodies (ACPA), which are present in a majority of RA patients, may be triggered in the gum mucosa. To address this hypothesis, we investigated the antibody response to a citrullinated P. gingivalis peptide in relation to the autoimmune ACPA response in early RA, and examined citrulline-reactivity in monoclonal antibodies derived from human gingival B cells. Antibodies to a citrullinated peptide derived from P. gingivalis (denoted CPP3) and human citrullinated peptides were analyzed by multiplex array in 2,807 RA patients and 372 controls; associations with RA risk factors and clinical features were examined. B cells from inflamed gingival tissue were single-cell sorted, and immunoglobulin (Ig) genes were amplified, sequenced, cloned and expressed (n=63) as recombinant monoclonal antibodies, and assayed for citrulline-reactivities by enzyme-linked immunosorbent assay. Additionally, affinity-purified polyclonal anti-cyclic-citrullinated peptide (CCP2) IgG, and monoclonal antibodies derived from RA blood and synovial fluid B cells (n=175), were screened for CPP3-reactivity. Elevated anti-CPP3 antibody levels were detected in RA (11%), mainly CCP2+ RA, compared to controls (2%), p<0.0001, with a significant association to HLA-DRB1 shared epitope alleles, smoking and baseline pain, but with low correlation to autoimmune ACPA fine-specificities. Monoclonal antibodies derived from gingival B cells showed cross-reactivity between P. gingivalis CPP3 and human citrullinated peptides, and a CPP3+/CCP2+ clone, derived from an RA blood memory B cell, was identified. Our data support the possibility that immunity to P. gingivalis derived citrullinated antigens, triggered in the inflamed gum mucosa, may contribute to the presence of ACPA in RA patients, through mechanisms of molecular mimicry.

Altered CXCR4 dynamics at the cell membrane impairs directed cell migration in WHIM syndrome patients.

Chemokine receptor nanoscale organization at the cell membrane is orchestrated by the actin cytoskeleton and influences cell responses. Using single-particle tracking analysis we show that CXCR4R334X, a truncated mutant chemokine receptor linked to WHIM syndrome (warts, hypogammaglobulinemia, infections, myelokathexis), fails to nanoclusterize after CXCL12 stimulation, and alters the lateral mobility and spatial organization of CXCR4 when coexpressed. These findings correlate with multiple phalloidin-positive protrusions in cells expressing CXCR4R334X, and their inability to correctly sense chemokine gradients. The underlying mechanisms involve inappropriate actin cytoskeleton remodeling due to the inadequate ß-arrestin1 activation by CXCR4R334X, which disrupts the equilibrium between activated and deactivated cofilin. Overall, we provide insights into the molecular mechanisms governing CXCR4 nanoclustering, signaling and cell function, and highlight the essential scaffold role of ß-arrestin1 to support CXCL12-mediated actin reorganization and receptor clustering. These defects associated with CXCR4R334X expression might contribute to the severe immunological symptoms associated with WHIM syndrome.

Narcolepsy: a model interaction between immune system, nervous system, and sleep-wake regulation.

Narcolepsy is a rare chronic neurological disorder characterized by an irresistible excessive daytime sleepiness and cataplexy. The disease is considered to be the result of the selective disruption of neuronal cells in the lateral hypothalamus expressing the neuropeptide hypocretin, which controls the sleep-wake cycle. Diagnosis and management of narcolepsy represent still a substantial medical challenge due to the large heterogeneity in the clinical manifestation of the disease as well as to the lack of understanding of the underlying pathophysiological mechanisms. However, significant advances have been made in the last years, thus opening new perspective in the field. This review describes the current knowledge of clinical presentation and pathology of narcolepsy as well as the existing diagnostic criteria and therapeutic intervention for the disease management. Recent evidence on the potential immune-mediated mechanisms that may underpin the disease establishment and progression are also highlighted.