Plasma cells are not restricted to the CD27+ phenotype: characterization of CD27-CD43+ antibody-secreting cells.

Circulating antibody-secreting cells are present in the peripheral blood of healthy individuals reflecting the continued activity of the humoral immune system. Antibody-secreting cells typically express CD27. Here we describe and characterize a small population of antibody-secreting class switched CD19+CD43+ B cells that lack expression of CD27 in the peripheral blood of healthy subjects. In this study, we characterized CD27-CD43+ cells. We demonstrate that class-switched CD27-CD43+ B cells possess characteristics of conventional plasmablasts as they spontaneously secrete antibodies, are morphologically similar to antibody-secreting cells, show downregulation of B cell differentiation markers, and have a gene expression profile related to conventional plasmablasts. Despite these similarities, we observed differences in IgA and IgG subclass distribution, expression of homing markers, replication history, frequency of somatic hypermutation, immunoglobulin repertoire, gene expression related to Toll-like receptors, cytokines, and cytokine receptors, and antibody response to vaccination. Their frequency is altered in immune-mediated disorders. Conclusion: we characterized CD27-CD43+ cells as antibody-secreting cells with differences in function and homing potential as compared to conventional CD27+ antibody-secreting cells.

Antinuclear antibodies in individuals with COVID-19 reflect underlying disease: Identification of new autoantibodies in systemic sclerosis (CDK9) and malignancy (RNF20, RCC1, TRIP13).

A high prevalence of antinuclear antibodies (ANA) in COVID-19 has been insinuated, but the nature of the target antigens is poorly understood. We studied ANA by indirect immunofluorescence in 229 individuals with COVID-19. The target antigens of high titer ANA (≥1:320) were determined by immunoprecipitation (IP) combined with liquid-chromatography-mass spectrometry (MS). High titer ANA (≥1:320) were found in 14 (6%) of the individuals with COVID-19. Of the 14 COVID-19 cases with high titer ANA, 6 had an underlying autoimmune disease and 5 a malignancy. IP-MS revealed known target antigens associated with autoimmune disease as well as novel autoantigens, including CDK9 (in systemic sclerosis) and RNF20, RCC1 and TRIP13 (in malignancy). The novel autoantigens were confirmed by IP-Western blotting. In conclusion, in depth analysis of the targets of high titer ANA revealed novel autoantigens in systemic sclerosis and in malignant disease.

Mass spectrometry-based identification of new anti-Ly and known antisynthetase autoantibodies.

OBJECTIVES: To discover new and detect known antisynthetase autoantibodies (ASAs) through protein immunoprecipitation combined with gel-free liquid chromatography-tandem mass spectrometry (IP-MS). METHODS: IP-MS was performed using sera of individuals showing features of antisynthetase syndrome (ASyS) without (n=5) and with (n=12) previously detected ASAs, and healthy controls (n=4). New candidate aminoacyl-tRNA-synthetase (ARS) autoantigens identified through unbiased IP-MS were confirmed by IP-western blot. A targeted IP-MS assay for various ASA specificities was developed and validated with sera of patients with known ASAs (n=16), disease controls (n=20) and healthy controls (n=25). The targeted IP-MS assay was applied in an additional cohort of patients with multiple ASyS features or isolated myositis without previously detected ASAs (n=26). RESULTS: Autoantibodies to cytoplasmic cysteinyl-tRNA-synthetase (CARS1) were identified by IP-MS and confirmed by western blot as a new ASA specificity, named anti-Ly, in the serum of a patient with ASyS features. Rare ASAs, such as anti-OJ, anti-Zo and anti-KS, and common ASAs could also be identified by IP-MS. A targeted IP-MS approach for ASA detection was developed and validated. Application of this method in an additional cohort identified an additional patient with anti-OJ autoantibodies that were missed by line and dot immunoassays. DISCUSSION: CARS1 is the dominant cognate ARS autoantigen of the newly discovered anti-Ly ASA specificity. Rare and common ASA specificities could be detected by both unbiased and targeted IP-MS. Unbiased and targeted IP-MS are promising methods for discovery and detection of autoantibodies, especially autoantibodies that target complex autoantigens.

Anti-Ki/anti-PA28γ autoantibodies contribute to the HEp-2 indirect immunofluorescence nuclear speckled pattern.

OBJECTIVES: Antinuclear antibodies (ANAs) are associated with several autoimmune diseases. Indirect immunofluorescence (IIF) on human epithelial type 2 (HEp-2) cells is the golden standard for ANA detection in the clinic. In case of a positive HEp-2 IIF test result, follow-up tests are done to determine autoantibody specificity. For a fraction of the HEp-2 IIF-positive samples, the nature of the autoantigens remains uncharacterized. Our objective was to characterize autoantigens in such samples. METHODS: To characterize autoantigens in an unbiased way, we combined protein immunoprecipitation with liquid chromatography (LC) tandem mass spectrometry (MS/MS) sequencing. RESULTS: Using such approach we detected the Ki antigen, also referred to as PA28γ, in the immunoprecipitate of serum samples of three individuals with an autoimmune disease. The HEp-2 nuclear speckled IIF fluorescent signal of all three serum samples was abolished after pre-absorption of the serum with recombinant Ki antigen, confirming that autoantibodies against Ki underlie the HEp-2 IIF signal. CONCLUSIONS: Our data suggest that anti-Ki autoantibodies can underlie a nuclear speckled HEp-2 IIF pattern.

Identification of SP1683 as a pneumococcal protein that is protective against nasopharyngeal colonization.

Serotype-independent protein-based pneumococcal vaccines represent attractive alternatives to capsular polysaccharide-based vaccines. The aim of this study was to identify novel immunogenic proteins from Streptococcus pneumoniae that may be used in protein-based pneumococcal vaccine. An immunoproteomics approach and a humanized severe combined immunodeficient mouse model were used to identify S. pneumoniae proteins that are immunogenic for the human immune system. Among the several proteins identified, SP1683 was selected, recombinantly produced, and infection and colonization murine models were used to evaluate the capacity of SP1683 to elicit protective responses, in comparison to known pneumococcal immunogenic proteins (PhtD and detoxified pneumolysin, dPly). Immunisation with SP1683 elicited a weaker antibody response than immunisation with PhtD and did not provide protection in the model of invasive disease. However, similar to PhtD, it was able to significantly reduce colonization in the mouse model of nasopharyngeal carriage. Treatment with anti-IL17A and anti-IL17F antibodies abolished the protection against colonization elicited by SP1683 or PhtD + dPly, which indicated that the protection afforded in this model was Th17-dependent. In conclusion, intranasal immunization with the pneumococcal protein SP1683 conferred IL17-dependent protection against nasopharyngeal carriage in mice, but systemic immunization did not protect against invasive disease. These results do not support the use of SP1683 as an isolated pneumococcal vaccine antigen. Nevertheless, SP1683 could be used as a first line of defence in formulations combining several proteins.

Functional Evaluation of an IKBKG Variant Suspected to Cause Immunodeficiency Without Ectodermal Dysplasia.

Hypomorphic IKBKG mutations in males are typically associated with anhidrotic ectodermal dysplasia with immunodeficiency (EDA-ID). Some mutations cause immunodeficiency without EDA (NEMO-ID). The immunological profile associated with these NEMO-ID variants is not fully documented. We present a 2-year-old patient with suspected immunodeficiency in which a hemizygous p.Glu57Lys IKBKG variant was identified. At the age of 1 year, he had an episode of otitis media that evolved into a bilateral mastoiditis (Pseudomonas spp). Hypogammaglobulinemia, specific (polysaccharide) antibody deficiency, and low switched memory B cell subsets were noticed. The mother was heterozygous for the variant but had no signs of incontinentia pigmenti. Patient peripheral blood mononuclear cells produced low amounts of IL-6 after stimulation with IL-1ß, Pam3CSK4, and FSL-1. In patient fibroblasts, IκB-α was degraded normally upon stimulation with IL-1ß or TNF-α. Transduction of wild-type and variant NEMO in NEMO-/- deficient SV40 fibroblasts revealed a slight but significant reduction of IL-6 production upon stimulation with IL-1ß and TNF-α. In conclusion, we demonstrated that p.Glu57Lys leads to specific immunological defects in vitro. No other pathogenic PID variants were identified through whole exome sequencing. As rare polymorphisms have been described in IKBKG and polygenic inheritance remains an option in the presented case, this study emphasizes the need for thorough functional and genetic evaluation when encountering and interpreting suspected disease-causing NEMO-ID variants.

PID in Disguise: Molecular Diagnosis of IRAK-4 Deficiency in an Adult Previously Misdiagnosed With Autosomal Dominant Hyper IgE Syndrome.

Autosomal recessive IL-1R-associated kinase 4 (IRAK-4) deficiency is a rare cause of recurrent pyogenic infections with limited inflammatory responses. We describe an adult female patient with severe lung disease who was phenotypically diagnosed as suffering from autosomal dominant Hyper IgE syndrome (AD HIES) because of recurrent skin infections with Staphylococcus aureus, recurrent pneumonia and elevated serum IgE levels. In contrast to findings in AD HIES patients, no abnormalities were found in the Th17 and circulating follicular helper T cell subsets. A panel-based sequencing approach led to the identification of a homozygous IRAK4 stop mutation (c.877C > T, p.Gln293*).

Anti-Pneumococcal Capsular Polysaccharide Antibody Response and CD5 B Lymphocyte Subsets.

The role of CD19(+) CD5(+) and CD19(+) CD5(-) B cell subpopulations in the antibody response to pneumococcal capsular polysaccharides (caps-PSs) is controversial. In the present study, we evaluated the role of human CD19(+) CD5(+) and CD19(+) CD5(-) cell populations in the serotype-specific antibody response to caps-PS. After vaccination of 5 healthy human adults with Pneumovax (23-valent pneumococcal polysaccharide vaccine [PPV23]), IgG anti-caps-PS serotype 4 antibody-producing cells resided mainly in the CD19(+) CD5(-) B cell subset, as assessed by enzyme-linked immunosorbent spot (ELISpot) analysis. Moreover, in a humanized SCID mouse model, CD19(+) CD5(-) B cells were more effective than CD19(+) CD5(+) cells in producing IgG anti-cap-PS antibodies. Finally, an association was found between the level of IgG anti-caps-PS antibodies and the number of CD19(+) CD5(-) B cells in 33 humans vaccinated with PPV23. Taken together, our data suggest that CD5 defines a functionally distinct population of B cells in humans in the anti-caps-PS immune response.

Addressing diagnostic challenges in primary immunodeficiencies: laboratory evaluation of Toll-like receptor- and NF-κB-mediated immune responses.

Toll-like receptors (TLRs) play an important role in immunity and mediate their actions via multiple signaling pathways, in particular, the nuclear factor of kappa light polypeptide gene enhancer in B-cells (NF-κB) pathway. Rare inherited defects of TLR- and NF-κB-dependent responses have recently been recognized. These primary immunodeficiencies predispose children to life-threatening infections and often remain undiagnosed. Establishing a sensitive, specific, cost-effective and simple method for diagnosis is therefore important. In this article, we review the known defects of TLR- and NF-κB-mediated pathways and the assays that can be used to screen for such defects.

Generation of antibody responses to pneumococcal capsular polysaccharides is independent of CD1 expression in mice.

Streptococcus pneumoniae is a bacterial microorganism that frequently causes serious infection, particularly in children and the elderly. Protection against infection with S. pneumoniae is based mainly on the generation of antibodies to the pneumococcal capsular polysaccharides (caps-PS), but the mechanisms responsible for the generation of anticapsular antibodies remain incompletely understood. The aim of the present study was to evaluate the role of CD1-restricted T cells in the antibody response to caps-PS. When immunized with Pneumo23, wild-type mice and CD1 knockout mice on BALB/c and C57BL/6 backgrounds generated immunoglobulin M (IgM) and IgG antibody responses to soluble caps-PS that were comparable. Similar results were obtained after immunization with heat-inactivated S. pneumoniae. The IgM and IgG antibody response of wild-type mice to Pneumo23 was not affected by an antagonizing monoclonal anti-CD1 antibody treatment. In summary, our data provide evidence that the antibody response to caps-PS is generated independently of CD1 expression.

Specific intracellular adhesion molecule-grabbing nonintegrin R1 is not involved in the murine antibody response to pneumococcal polysaccharides.

Streptococcus pneumoniae is a microorganism that frequently causes serious infections in children, the elderly, and immunocompromised patients. We studied whether the specific intracellular adhesion molecule-grabbing nonintegrin R1 (Sign-R1) receptor, involved in the uptake of capsular polysaccharides (caps-PS) by antigen-presenting cells, is necessary for the antibody response to pneumococcal caps-PS and phosphorylcholine (PC). The antibody response to caps-PS and PC was evaluated after vaccination with soluble caps-PS (Pneumovax) and after vaccination with heat-killed S. pneumoniae. The role of Sign-R1 was investigated by using Sign-R1 knockout mice and anti-Sign-R1 monoclonal antibodies. The immunoglobulin M (IgM) and IgG antibody response to PC and caps-PS (serotypes 3 and 14) was not affected by anti-Sign-R1 monoclonal antibodies. The IgM antibody response in Sign-R1 knockout mice was comparable to the antibody response in wild-type mice. The IgG antibody response to serotype 3, but not to serotype 14, tended to be lower in Sign-R1 knockout mice compared to wild-type mice. In conclusion, we found that Sign-R1 is not involved in the IgM antibody production to PC and caps-PS serotype 3 or 14 and the IgG immune response to PC and caps-PS serotype 14. There is no direct relation between capture and uptake of caps-PS serotype 14 by Sign-R1 and the initiation of the anti-caps-PS antibody production in mice.

Distinct approaches to investigate the importance of the murine 4-1BB 4-1BBL interaction in the antibody response to Streptococcus pneumoniae.

Protection against infection with Streptococcus pneumoniae is based mainly on the generation of antibodies to the pneumococcal capsular polysaccharides (caps-PS). Although caps-PS are considered thymus-independent antigens, there is a growing body of evidence that T lymphocytes and costimulatory molecules are involved in the regulation of the antibody response to caps-PS. We investigated whether the interaction between 4-1BB and 4-1BB ligand (4-1BBL) is involved in the modulation of the antibody response to caps-PS after immunization with Pneumovax or with intact heat-killed S. pneumoniae. Treatment with agonistic anti-4-1BB mAb, which mimics engagement of 4-1BB by 4-1BBL, had no effect on the IgG and IgM immune response to caps-PS (Serotype 3) after immunization with Pneumovax or with S. pneumoniae Serotype 3. However, anti-4-1BB treatment strongly inhibited the IgG response to pneumococcal surface protein A (PspA). By contrast, the IgG anti-caps-PS (Serotype 3) antibody response was reduced strongly in 4-1BBL(-/-) mice immunized with S. pneumoniae Serotype 3. The IgG anti-PspA antibody response in the 4-1BB(-/-) mice was comparable with the immune response in the wild-type mice. We conclude that distinct pathways are involved in the humoral antibody response to pneumococcal antigens, depending on the nature of the antigen and the context in which the different antigens are presented. The 4-1BB-4-1BBL interaction is not involved in the antibody response to soluble caps-PS. The influence of the 4-1BB-4-1BBL interaction in the immune reaction to S. pneumoniae Serotype 3 depends on the experimental system used.

Laboratory diagnosis of specific antibody deficiency to pneumococcal capsular polysaccharide antigens.

BACKGROUND: Measurement of postimmunization antibody response to pneumococcal capsular polysaccharide (caps-PS) is the standard method to identify deficiency of antipolysaccharide antibody production. However, no standardized criteria have been defined for classification of patients into responders or nonresponders to caps-PS. METHODS: We vaccinated 37 healthy children and 39 healthy adults with Pneumovax and measured the anti-caps-PS antibody response to 5 serotypes. We also measured antipneumococcal antibody titers in 82 patients with increased susceptibility to airway infection. The ELISA was performed according to the 3rd-generation assay format. RESULTS: The lower 5th percentile (cutoff) concentrations for the postimmunization antibody titer in healthy individuals were 0.67 mg/L, 0.45 mg/L, 0.46 mg/L, 0.31 mg/L, and 1.04 mg/L for serotypes 3, 4, 9N, 18C, and 19F, respectively. In 96% of healthy individuals, antibody responses higher than the cutoff concentration were seen for at least 3 of the 5 serotypes. Nine of 82 patients (11%) failed to mount an adequate antibody response for at least 4 of the 5 serotypes tested, whereas only 1 control (1.3%) failed to do so. CONCLUSION: The cutoffs for antibody responses to caps-PS identified in this study appear useful for identifying individuals with an inadequate response to vaccine.

CD4+ T lymphocytes expressing CD40 ligand help the IgM antibody response to soluble pneumococcal polysaccharides via an intermediate cell type.

Streptococcus pneumoniae causes serious infections in children, the elderly, and immunocompromised patients. Protection against infections with S. pneumoniae is mediated through Abs against the capsular polysaccharides (caps-PS). We previously showed that the murine Ab response to caps-PS is dependent on CD40-CD40L interaction. In the present paper, we addressed the question of whether the CD40-CD40L-mediated modulation of the anti-caps-PS immune reaction is the result of a direct interaction between B lymphocytes and T lymphocytes or of an indirect interaction. SCID/SCID mice reconstituted with B lymphocytes from wild-type mice did not mount anti-caps-PS Abs. SCID/SCID mice reconstituted with B lymphocytes from wild-type mice and CD4+ T lymphocytes from wild-type mice but not CD4+ T lymphocytes from CD40L knockout mice stimulated the anti-caps-PS Ab response. This indicated that CD4+ T lymphocytes stimulated the anti-caps-PS Ab response in a CD40L-dependent manner. SCID/SCID mice reconstituted with B lymphocytes from CD40 knockout mice and CD4+ T lymphocytes from wild-type mice generated an anti-caps-PS Ab response that could be inhibited by MR1, a blocking anti-CD40L Ab. These data indicated that CD4+ T lymphocytes stimulated the anti-caps-PS Ab response in an indirect way. Finally, lethally irradiated CD40 knockout mice reconstituted with bone marrow from wild-type mice mounted an anti-caps-PS Ab response that was comparable to the Ab response in wild-type mice, revealing that the required CD40 was on hemopoietic cells. In conclusion, we provide evidence that CD4+ T lymphocytes expressing CD40L stimulate the Ab response to soluble caps-PS by interacting with CD40-expressing non-B cells.