Malaria-driven expansion of adaptive-like functional CD56-negative NK cells correlates with clinical immunity to malaria.

Natural killer (NK) cells likely play an important role in immunity to malaria, but the effect of repeated malaria on NK cell responses remains unclear. Here, we comprehensively profiled the NK cell response in a cohort of 264 Ugandan children. Repeated malaria exposure was associated with expansion of an atypical, CD56neg population of NK cells that differed transcriptionally, epigenetically, and phenotypically from CD56dim NK cells, including decreased expression of PLZF and the Fc receptor γ-chain, increased histone methylation, and increased protein expression of LAG-3, KIR, and LILRB1. CD56neg NK cells were highly functional and displayed greater antibody-dependent cellular cytotoxicity than CD56dim NK cells. Higher frequencies of CD56neg NK cells were associated with protection against symptomatic malaria and high parasite densities. After marked reductions in malaria transmission, frequencies of these cells rapidly declined, suggesting that continuous exposure to Plasmodium falciparum is required to maintain this modified, adaptive-like NK cell subset.

Clonal replacement sustains long-lived germinal centers primed by respiratory viruses.

Germinal centers (GCs) form in secondary lymphoid organs in response to infection and immunization and are the source of affinity-matured B cells. The duration of GC reactions spans a wide range, and long-lasting GCs (LLGCs) are potentially a source of highly mutated B cells. We show that rather than consisting of continuously evolving B cell clones, LLGCs elicited by influenza virus or SARS-CoV-2 infection in mice are sustained by progressive replacement of founder clones by naive-derived invader B cells that do not detectably bind viral antigens. Rare founder clones that resist replacement for long periods are enriched in clones with heavily mutated immunoglobulins, including some with very high affinity for antigen, that can be recalled by boosting. Our findings reveal underappreciated aspects of the biology of LLGCs generated by respiratory virus infection and identify clonal replacement as a potential constraint on the development of highly mutated antibodies within these structures.

Increases in ambient air pollutants during pregnancy are linked to increases in methylation of IL4, IL10, and IFNγ.

BACKGROUND: Ambient air pollutant (AAP) exposure is associated with adverse pregnancy outcomes, such as preeclampsia, preterm labor, and low birth weight. Previous studies have shown methylation of immune genes associate with exposure to air pollutants in pregnant women, but the cell-mediated response in the context of typical pregnancy cell alterations has not been investigated. Pregnancy causes attenuation in cell-mediated immunity with alterations in the Th1/Th2/Th17/Treg environment, contributing to maternal susceptibility. We recruited women (n = 186) who were 20 weeks pregnant from Fresno, CA, an area with chronically elevated AAP levels. Associations of average pollution concentration estimates for 1 week, 1 month, 3 months, and 6 months prior to blood draw were associated with Th cell subset (Th1, Th2, Th17, and Treg) percentages and methylation of CpG sites (IL4, IL10, IFNγ, and FoxP3). Linear regression models were adjusted for weight, age, season, race, and asthma, using a Q value as the false-discovery-rate-adjusted p-value across all genes. RESULTS: Short-term and mid-term AAP exposures to fine particulate matter (PM2.5), nitrogen dioxide (NO2) carbon monoxide (CO), and polycyclic aromatic hydrocarbons (PAH456) were associated with percentages of immune cells. A decrease in Th1 cell percentage was negatively associated with PM2.5 (1 mo/3 mo: Q < 0.05), NO2 (1 mo/3 mo/6 mo: Q < 0.05), and PAH456 (1 week/1 mo/3 mo: Q < 0.05). Th2 cell percentages were negatively associated with PM2.5 (1 week/1 mo/3 mo/6 mo: Q < 0.06), and NO2 (1 week/1 mo/3 mo/6 mo: Q < 0.06). Th17 cell percentage was negatively associated with NO2 (3 mo/6 mo: Q < 0.01), CO (1 week/1 mo: Q < 0.1), PM2.5 (3 mo/6 mo: Q < 0.05), and PAH456 (1 mo/3 mo/6 mo: Q < 0.08). Methylation of the IL10 gene was positively associated with CO (1 week/1 mo/3 mo: Q < 0.01), NO2 (1 mo/3 mo/6 mo: Q < 0.08), PAH456 (1 week/1 mo/3 mo: Q < 0.01), and PM2.5 (3 mo: Q = 0.06) while IL4 gene methylation was positively associated with concentrations of CO (1 week/1 mo/3 mo/6 mo: Q < 0.09). Also, IFNγ gene methylation was positively associated with CO (1 week/1 mo/3 mo: Q < 0.05) and PAH456 (1 week/1 mo/3 mo: Q < 0.06). CONCLUSION: Exposure to several AAPs was negatively associated with T-helper subsets involved in pro-inflammatory and anti-inflammatory responses during pregnancy. Methylation of IL4, IL10, and IFNγ genes with pollution exposure confirms previous research. These results offer insights into the detrimental effects of air pollution during pregnancy, the demand for more epigenetic studies, and mitigation strategies to decrease pollution exposure during pregnancy.

Immune Dysregulation in Acute SARS-CoV-2 Infection.

Introduction: Neutralizing antibodies have been shown to develop rapidly following SARS-CoV-2 infection, specifically against spike (S) protein, where cytokine release and production is understood to drive the humoral immune response during acute infection. Thus, we evaluated the quantity and function of antibodies across disease severities and analyzed the associated inflammatory and coagulation pathways to identify acute markers that correlate with antibody response following infection. Methods: Blood samples were collected from patients at time of diagnostic SARS-CoV-2 PCR testing between March 2020-November 2020. Plasma samples were analyzed using the MesoScale Discovery (MSD) Platform using the COVID-19 Serology Kit and U-Plex 8 analyte multiplex plate to measure anti-alpha and beta coronavirus antibody concentration and ACE2 blocking function, as well as plasma cytokines. Results: A total of 230 (181 unique patients) samples were analyzed across the 5 COVID-19 disease severities. We found that antibody quantity directly correlated with functional ability to block virus binding to membrane-bound ACE2, where a lower SARS-CoV-2 anti-spike/anti-RBD response corresponded with a lower antibody blocking potential compared to higher antibody response (anti-S1 r = 0.884, P < 0.001; anti-RBD r = 0.75, P < 0.001). Across all the soluble proinflammatory markers we examined, ICAM, IL-1ß, IL-4, IL-6, TNFα, and Syndecan showed a statistically significant positive correlation between cytokine or epithelial marker and antibody quantity regardless of COVID-19 disease severity. Analysis of autoantibodies against type 1 interferon was not shown to be statistically significant between disease severity groups. Conclusion: Previous studies have shown that proinflammatory markers, including IL-6, IL-8, IL-1ß, and TNFα, are significant predictors of COVID-19 disease severity, regardless of demographics or comorbidities. Our study demonstrated that not only are these proinflammatory markers, as well as IL-4, ICAM, and Syndecan, correlative of disease severity, they are also correlative of antibody quantity and quality following SARS-CoV-2 exposure.

New-Onset IgG Autoantibodies in Hospitalized Patients with COVID-19.

Coronavirus Disease 2019 (COVID-19), caused by Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), is associated with a wide range of clinical manifestations, including autoimmune features and autoantibody production. We developed three different protein arrays to measure hallmark IgG autoantibodies associated with Connective Tissue Diseases (CTDs), Anti-Cytokine Antibodies (ACA), and anti-viral antibody responses in 147 hospitalized COVID-19 patients in three different centers. Autoantibodies were identified in approximately 50% of patients, but in <15% of healthy controls. When present, autoantibodies largely targeted autoantigens associated with rare disorders such as myositis, systemic sclerosis and CTD overlap syndromes. Anti-nuclear antibodies (ANA) were observed in ∼25% of patients. Patients with autoantibodies tended to demonstrate one or a few specificities whereas ACA were even more prevalent, and patients often had antibodies to multiple cytokines. Rare patients were identified with IgG antibodies against angiotensin converting enzyme-2 (ACE-2). A subset of autoantibodies and ACA developed de novo following SARS-CoV-2 infection while others were transient. Autoantibodies tracked with longitudinal development of IgG antibodies that recognized SARS-CoV-2 structural proteins such as S1, S2, M, N and a subset of non-structural proteins, but not proteins from influenza, seasonal coronaviruses or other pathogenic viruses. COVID-19 patients with one or more autoantibodies tended to have higher levels of antibodies against SARS-CoV-2 Nonstructural Protein 1 (NSP1) and Methyltransferase (ME). We conclude that SARS-CoV-2 causes development of new-onset IgG autoantibodies in a significant proportion of hospitalized COVID-19 patients and are positively correlated with immune responses to SARS-CoV-2 proteins.

Suppression of autoimmunity via microbial mimics of altered peptide ligands.

Molecular mimics of self-antigens can behave as altered peptide ligands and serve to ameliorate autoimmune disease. Analysis of experimental autoimmune encephalomyelitis with proteomic autoantibody microarrays reveals that there might exist a wide variety of microbes with features that mimic self-epitopes. Autoimmunity could therefore be modulated via microbial immunity, which may account for relapse and remission of ongoing disease.

Detection of apoptosis-specific autoantibodies directed against granzyme B-induced cleavage fragments of the SS-B (La) autoantigen in sera from patients with primary Sjögren's syndrome.

The objective of this study was to detect autoantibodies against granzyme B cleavage products in sera from patients with primary Sjögren's syndrome (SS). Cell lysates derived from human salivary gland (HSG) cell lines were incubated with granzyme B. The susceptibility to the generation of cleavage fragments of SS autoantigens was assayed by immunoblotting using sera from 57 primary SS patients, 17 primary SS patients with malignant lymphoma (ML), 28 systemic lupus erythematosus (SLE) patients, and 20 healthy controls. A 27 kD protein was recognized by serum autoantibodies in 8 (14.0%) of 57 primary SS patients, 5 (29.4%) of 17 SS patients with ML, 2 (7.1%) of 28 SLE patients, but not in 20 normal subjects. This protein was recognized by anti-SSB (La) monoclonal antibodies. Granzyme B-treated recombinant La protein was also shown to migrate as a discrete 27 kD protein by SDS PAGE. Blocking studies demonstrated the existence of an apoptosis-specific B cell epitope present in sera from 2 of 8 primary SS patients and in 2 of 5 primary SS patients with ML which recognized the 27 kD protein. Granzyme B-induced La fragments are generated during cytotoxicity in vitro. This is the first report describing autoantibodies in sera from primary SS patients that specifically recognize fragments of the La protein that are produced by the granzyme B protease.

Multiplexed assays for identification of biomarkers and surrogate markers in systemic lupus erythematosus.

Validated biomarkers and surrogate markers are badly needed for monitoring patients with systemic lupus erythematosus (SLE), both for routine clinical care and for clinical trials research. SLE is difficult to study in clinical trials, in part because the disease is so heterogeneous. Very few useful markers have been identified, and even those that historically have been thought to be valid have been recently questioned. This report will focus on the use of emerging multiplexed assay formats that enable analysis of hundreds or even thousands of analytes simultaneously. Their potential and pitfalls for monitoring patients with SLE, particularly those enrolled in clinical trials testing novel therapeutics, will be discussed.

Autoantibodies in early arthritis: advances in diagnosis and prognostication.

Several excellent reviews have recently been published on the significance of autoantibodies in rheumatoid arthritis (RA) (1-4). Here we: (i) review selected longitudinal studies examining the predictive utility of autoantibodies in early arthritis and early RA cohorts; (ii) assess the relevance of autoantibodies as an independent parameter for prediction and prognostication of RA; and (iii) describe the potential of multiplex autoantibody assays, including miniaturized, high-throughput microarray technology, to improve diagnosis and prognostication in recent-onset synovitis/early arthritis patients.

21st European Workshop for Rheumatology Research, Vienna, Austria, 1-4 March 2001.

Major advances in technology now drive how we approach questions in immunology, particularly in analyzing complex data sets commonly encountered in genomics and proteomics studies. Active areas of investigation include development of novel technologies, identification of elusive target antigens for RA and other diseases, dissection of signaling pathways connecting the lymphocyte cell surface with the nucleus, and exploration of new avenues for therapeutic interventions. The European Workshop for Rheumatology Research (EWRR) is a forum for many European and non-European scientists to present research findings of high quality. Arthritis researchers from around the globe should be strongly encouraged to attend future meetings, the next of which is the 22nd EWRR meeting in Leiden, the Netherlands, in 2002.

Monoclonal antibodies derived from BALB/c mice immunized with apoptotic Jurkat T cells recognize known autoantigens.

It has been postulated that post-translational modifications and relocalization of proteins during apoptosis may lead to presentation of these molecules to the immune system in such a way that normal mechanisms of tolerance are bypassed. In the present study, Jurkat cells were induced to undergo apoptosis by treatment with the chemotherapeutic agent Ara-C. BALB/c mice were then immunized with the apoptotic cells and hybridomas were generated. Using an indirect immunofluorescence assay, the monoclonal antibodies produced were screened by flow cytometry for those monoclonal antibodies demonstrating reactivity with permeabilized apoptotic Jurkat cells but not with non-permeabilized normal or apoptotic Jurkat cells. Of 281 monoclonal antibodies, 20 monoclonal antibodies with these properties were selected for further analysis. Using 32P- or 35S-metabolically labelled Jurkat cells, these selected monoclonal antibodies were screened for their ability to recognize autoantigens by immunoprecipitation and Western blotting. Well characterized autoimmune sera were then used to confirm the identity of autoantigens by immunoblotting. We demonstrate that immunization of normal mice with apoptotic Jurkat cells results in the formation of antibodies targeting multiple autoantigens or autoantigen complexes, including Ku, rRNPs, snRNPs and vimentin. These findings are consistent with the hypothesis that apoptosis can contribute to the development of autoimmunity.

Death, autoantigen modifications, and tolerance.

Autoantibodies present in the serum of patients with a variety of inflammatory diseases have proven useful as diagnostic markers and as probes with which to elucidate biochemical and signaling pathways. The mechanisms governing the generation of autoantibodies remain elusive, constituting a critical missing link in our understanding of rheumatologic illnesses. Several lines of experimentation in recent years have strongly implicated events surrounding cell death in this process. This review will address the potential role played by death-specific modifications of autoantigens in bypassing tolerance to highly conserved autoantigens, including nucleic acids, lipids, and proteins.

Life and death decisions: regulation of apoptosis by proteolysis of signaling molecules.

Caspases are the major executioners of cell death, serving as molecular guillotines to behead many proteins required for maintenance of cellular homeostasis. Identification of caspase substrates has taken on increasing importance as we attempt to better understand the molecular mechanisms involved in regulating the struggle between life and death. Many caspase substrates have been described and include RNA binding proteins such as La and U1-70 kD, structural proteins such as keratin and nuclear lamins, and transcription factors or their regulatory proteins that include IkappaB, SP1, and SREBP. Kinases and other signaling proteins are perfectly suited to regulate life and death decisions in response to cellular stressors and have only recently been identified as important caspase substrates. Here we review the current status of signaling pathways that are activated, inactivated or dysregulated by proteases such as caspases and calpain to control entry into apoptosis. The emerging concept that some caspase pathways may be inhibited by cellular and viral apoptosis inhibitory proteins while other caspase pathways are preserved suggests that a subset of these kinases may exist as cleaved 'isoforms' in cells that are not destined to perish. By acting as executioners and as important 'molecular sensors' of the degree of cellular injury, the signaling proteins described in this review are strong candidates to mediate downstream events, both in condemned and in viable cells.

Small nucleolar RNP scleroderma autoantigens associate with phosphorylated serine/arginine splicing factors during apoptosis.

OBJECTIVE: Proteins that are phosphorylated during apoptosis are commonly precipitated by autoantibodies found in the sera of patients with systemic lupus erythematosus. We sought to determine whether scleroderma autoantigens such as small nucleolar RNPs (snoRNP) also associate with phosphoproteins in response to various cellular stressors. METHODS: We screened a panel of monoclonal antibodies derived from mice exposed to mercury, a well-characterized murine model of the anti-snoRNP autoimmune response, for the ability to selectively precipitate phosphoproteins from radiolabeled lysates prepared from Jurkat T cells subjected to stressful stimuli. RESULTS: Monoclonal antibodies reactive with snoRNPs precipitated a phosphoprotein complex (pp42, pp34, and pp23) from lysates prepared from apoptotic cells. Several novel phosphoproteins (pp62 and pp18) were also observed. The phosphorylation and/or recruitment of these proteins to the snoRNP complex is induced by multiple apoptotic stimuli (e.g., Fas ligation, anisomycin, or ultraviolet irradiation), an effect that is blocked by overexpression of Bcl-2. We were unable to demonstrate an association of the phosphoprotein complex with snoRNPs in cells treated with the xenobiotic agent mercury. The snoRNP-associated phosphoprotein complex is composed of serine/arginine (SR) splicing factors, including SRp40. CONCLUSION: The association of phosphorylated SR proteins with snoRNPs in cells undergoing apoptosis suggests that the immune response to fibrillarin that characterizes a subset of patients with scleroderma may be related to cell death induced by apoptotic stimuli (e.g., Fas ligation, irradiation, or chemical toxins), or by exposure to mercury.

The fate of U1 snRNP during anti-Fas induced apoptosis: specific cleavage of the U1 snRNA molecule.

During apoptosis, the U1-70K protein, a component of the spliceosomal U1 snRNP complex, is specifically cleaved by the enzyme caspase-3, converting it into a C-terminally truncated 40-kDa fragment. In this study, we show that the 40-kDa U1-70K fragment is still associated with the complete U1 snRNP complex, and that no obvious modifications occur with the U1 snRNP associated proteins U1A, U1C and Sm-B/B'. Furthermore, it is described for the first time that the U1 snRNA molecule, which is the backbone of the U1 snRNP complex, is modified during apoptosis by the specific removal of the first 5 - 6 nucleotides including the 2,2, 7-trimethylguanosine (TMG) cap. The observations that U1 snRNA cleavage is very specific (no such modifications were detected for the other U snRNAs tested) and that U1 snRNA cleavage is markedly inhibited in the presence of caspase inhibitors, indicate that an apoptotically activated ribonuclease is responsible for the specific modification of U1 snRNA during apoptosis.

The La (SS-B) autoantigen, a key protein in RNA biogenesis, is dephosphorylated and cleaved early during apoptosis.

In the past few years, a role for apoptotic processes in the development of autoimmune diseases has been suggested. An increasing number of cellular proteins, which are modified during apoptosis, has been described, and many of these proteins have been identified as autoantigens. We have studied the effects of apoptosis on the La protein in more detail and for the first time demonstrate that this autoantigen is rapidly dephosphorylated after the induction of apoptosis. Dephosphorylation of the La protein was observed after induction of apoptosis by several initiators and in various cell types. Furthermore, we demonstrate that at least a subset of the La protein is proteolytically cleaved in vivo, generating a 45 kDa fragment. Dephosphorylation as well as cleavage of La is inhibited by ZnSO4 as well as by several tetrapeptide caspase inhibitors, indicating that these processes require the activation of caspases. Dephosphorylation of La is inhibited by low concentrations of okadaic acid, suggesting that a PP2A-like phosphatase is involved. Generation of the 45 kDa fragment is consistent with proteolytic cleavage at amino acids 371 and/or 374. The possible significance of the apoptotic changes in the La protein for autoantibody production is discussed.

Rapid nucleolytic degradation of the small cytoplasmic Y RNAs during apoptosis.

We have investigated the fate of the RNA components of small ribonucleoprotein particles in apoptotic cells. We show that the cytoplasmic Ro ribonucleoprotein-associated Y RNAs are specifically and rapidly degraded during apoptosis via a caspase-dependent mechanism. This is the first study describing the selective degradation of a specific class of small structural RNA molecules in apoptotic cells. Cleavage and subsequent truncation of Y RNAs was observed upon exposure of cells to a variety of apoptotic stimuli and were found to be inhibited by Bcl-2, zinc, and several caspase inhibitors. These results indicate that apoptotic degradation of Y RNAs is dependent on caspase activation, which suggests that the nucleolytic activity responsible for hY RNA degradation is activated downstream of the caspase cascade. The Y RNA degradation products remain bound by the Ro60 protein and in part also by the La protein, the only two proteins known to be stably associated with intact Ro ribonucleoprotein particles. The size of the Y RNA degradation products is consistent with the protection from degradation of the most highly conserved region of the Y RNAs by the bound Ro60 and La proteins. Our results indicate that the rapid abrogation of the yet unknown function of Y RNAs might be an early step in the systemic deactivation of the dying cell.

The 72-kDa component of signal recognition particle is cleaved during apoptosis.

Proteins cleaved by apoptotic caspases are commonly recognized by autoantibodies found in the serum of patients with rheumatic disease. We report that the 72-kDa signal recognition particle (SRP) protein, a rare target of autoantibodies found in the serum of patients with dermatomyositis and systemic lupus erythematosus, is rapidly cleaved in Jurkat T cells treated with apoptotic (i.e. Fas ligation, treatment with gamma or ultraviolet radiation, or co-culture with anisomycin or staurosporine) but not proliferative (CD3 cross-linking) stimuli. Cleavage of SRP 72 produces a 66-kDa amino-terminal fragment and a 6-kDa carboxyl-terminal fragment that is selectively phosphorylated on serine residues. Cleavage of SRP 72 is prevented by chemical and peptide caspase inhibitors, and by overexpression of bcl-2, an inhibitor of apoptotic cell death. Analysis of the carboxyl terminus of SRP 72 has identified a putative cleavage site (SELD/A) for group III caspases, and carboxyl-terminal serine residues that are highly conserved in phylogeny. Both serine phosphorylation and caspase cleavage of SRP 72 are observed in cells derived from human, dog, rat, and mouse. Canine SRP 72 is cleaved in vitro by recombinant caspase 3 but retains the ability to mediate transport of a signal peptide-containing protein into the endoplasmic reticulum lumen. The 72-kDa component of the SRP joins a growing list of autoantigens that undergo post-translational modifications during programmed cell death.