Ig Isotypes Research Articles

Fc properties of SARS-CoV-2 spike-specific antibodies elicited by vaccines and infection

Abstract Since its emergence in 2019, SARS-CoV-2 has infected millions of people and several vaccines have been approved for clinical use worldwide. Antibodies against the spike protein of SARS-CoV-2 are a critical immune determinant for protection against this virus. While virus neutralization is a key function of spike-specific antibodies, antibodies also mediate Fc-dependent activities that can play a role in protection or pathogenesis. This study compared the Fc properties and functions of anti-spike antibodies elicited by Pfizer/BioNtech or Moderna mRNA vaccines, Sputnik rAd26+rAd5 vaccines, or natural SARS-CoV-2 infection. We first examined immunoglobulin (Ig) isotypes in sera or plasma from the three groups. IgG1 antibodies against the spike protein were the predominant isotype, produced to high levels by the vast majority of the subjects in the three groups. In addition to IgG1, the mRNA vaccines also elicited IgM, IgG2-4, and IgA1-2, whereas the Sputnik vaccines induced only IgG4. Convalescent COVID-19 patients had yet a distinct set of isotypes, displaying mainly IgM and IgA1 alongside IgG1. When we examined antibody functions, spike-specific antibodies from both mRNA and Sputnik vaccine groups showed greater capacities to bind and activate complement than those from the convalescent COVID-19 patients. However, the Sputnik vaccine-induced antibodies had a greater potency of binding the ADCC-activating FcγRIIIa receptor, although the Sputnik vaccines induced lower Ig levels against spike than the mRNA vaccines. These data demonstrate that antibodies with distinct Ig isotypes/subtypes and Fc functions were elicited by different COVID-19 vaccines and by natural SARS-CoV-2 infection. This work was supported in part by the Department of Veterans Affairs [Merit Review Grant I01BX005794] (C.E.H.) and [Research Career Scientist Award 1IK6BX004607] (C.E.H.); the National Institutes of Health [grant AI139290] to C.E.H., [grants R01 AI123449, R21 AI1498033] to B.L, the Department of Medicine of the Icahn School of Medicine at Mount Sinai (C.E.H.); the Department of Microbiology and the Ward-Coleman estate for endowing the Ward-Coleman Chairs at the Icahn School of Medicine at Mount Sinai (B.L.), and institutional funding as well as anonymous philanthropic donations (V.S.).

Mechanisms of TRAF3 regulation of TLR signaling in B lymphocytes

Abstract Toll-like receptors (TLRs) are pattern recognition receptors that bridge the innate and adaptive immune system and are critical for host defense. TLR signaling to B cells is important in normal immunity, but also implicated in autoimmune disease and B cell lymphomas (BCL). Most studies focus on the role of TLRs in myeloid cell populations. However, B cells express most TLRs and are highly responsive to TLR ligands, yet TLR-mediated B cell pathways are understudied. Previous studies from our lab show that TRAF3 inhibits B cell TLR functions, including cytokine production, Ig isotype switching, and antibody production in response to ligands for TLRs 3, 4, 7/8 and 9. A key knowledge gapis identifying the molecular mechanisms by which TRAF3 mediates this regulation. Our working hypothesisis that B cell-TRAF3 regulates the recruitment and/or post-translational modification of specific TLR complex signaling proteins. Our recent data show that B cell-TRAF3 forms a heterocomplex with TRAF6, associating with the tyrosine kinase Syk. In the absence of TRAF3, TRAF6 shows a greater association with several TLR signaling proteins, including MyD88, IRAK1 and Syk. This suggests that TRAF3 may inhibit TRAF6 access to the TLR signaling complex, and thus early TLR signaling. In addition, our work introduces a role for Syk in TLR signaling in B cells. In the absence of TRAF3, activation of Syk is enhanced in response to ligands for TLRs 4 and 7. Inhibition of Syk activation also revealed downstream effects on TLR-mediated NFκB activation. Further studies aim at providing new insights into molecular mechanisms contributing to the regulation of B cell-TRAF3 in TLR signaling. VA Merit Review I01 BX001702 NIH P50 CA97274 RO1 AI5107312000000113259002001 NIH T32 Training Grant AI07485 RO1 AI1762656

Effects of IL-9 on TRAF3-deficient B cell activation

Abstract The pleiotropic cytokine interleukin-9 (IL-9) signals to target cells by binding to a heterodimeric receptor consisting of the IL-9 receptor (IL-9R) and the common subunit γ chain. In the present study, we found that the expression of IL-9R was strikingly up-regulated in mouse naïve follicular B cells genetically deficient in TRAF3, a critical regulator of B cell survival and function. The highly up-regulated IL-9R on Traf3 −/−follicular B cells conferred responsiveness to IL-9, including IgM production and STAT3 phosphorylation. Interestingly, IL-9 significantly enhanced class switch recombination to IgG1 induced by B cell antigen receptor crosslinking plus IL-4 in Traf3 −/−B cells but not in wild type B cells. Blocking the JAK-STAT3 pathway abrogated the effects of IL-9 on Traf3 −/−B cells. Our study thus revealed a novel pathway that TRAF3 suppresses B cell activation and Ig isotype switching by inhibiting IL-9R-JAK-STAT3 signaling. Our findings provide new insights into the TRAF3-IL-9R axis in B cell function and have significant implications for the understanding and treatment of a variety of human diseases involving aberrant B cell activation. This study was supported by the National Institutes of Health grants R01 CA158402 (P. Xie) and R21 AI128264 (P. Xie), the Department of Defense grant W81XWH-13-1-0242 (P. Xie), and a Busch Biomedical Grant (P. Xie). FACS analyses were supported by the Flow Cytometry Core Facility with funding from NCI-CCSG P30CA072720.

CLL stereotyped B-cell receptor immunoglobulin sequences are recurrent in the B-cell repertoire of healthy individuals: Apparent lack of central and early peripheral tolerance censoring.

The leukemic cells of patients with chronic lymphocytic leukemia (CLL) are often unique, expressing remarkably similar IGHV-IGHD-IGHJ gene rearrangements, "stereotyped BCRs". The B-cell receptors (BCRs) on CLL cells are also distinctive in often deriving from autoreactive B lymphocytes, leading to the assumption of a defect in immune tolerance. Using bulk and single-cell immunoglobulin heavy and light chain variable domain sequencing, we enumerated CLL stereotype-like IGHV-IGHD-IGHJ sequences (CLL-SLS) in B cells from cord blood (CB) and adult peripheral blood (PBMC) and bone marrow (BM of healthy donors. CLL-SLS were found at similar frequencies among CB, BM, and PBMC, suggesting that age does not influence CLL-SLS levels. Moreover, the frequencies of CLL-SLS did not differ among B lymphocytes in the BM at early stages of development, and only re-circulating marginal zone B cells contained significantly higher CLL-SLS frequencies than other mature B-cell subpopulations. Although we identified CLL-SLS corresponding to most of the CLL major stereotyped subsets, CLL-SLS frequencies did not correlate with those found in patients. Interestingly, in CB samples, half of the CLL-SLS identified were attributed to two IGHV-mutated subsets. We also found satellite CLL-SLS among the same normal samples, and they were also enriched in naïve B cells but unexpectedly, these were ~10-fold higher than standard CLL-SLS. In general, IGHV-mutated CLL-SLS subsets were enriched among antigen-experienced B-cell subpopulations, and IGHV-unmutated CLL-SLS were found mostly in antigen-inexperienced B cells. Nevertheless, CLL-SLS with an IGHV-mutation status matching that of CLL clones varied among the normal B-cell subpopulations, suggesting that specific CLL-SLS could originate from distinct subpopulations of normal B cells. Lastly, using single-cell DNA sequencing, we identified paired IGH and IGL rearrangements in normal B lymphocytes resembling those of stereotyped BCRs in CLL, although some differed from those in patients based on IG isotype or somatic mutation. CLL-SLS are present in normal B-lymphocyte populations at all stages of development. Thus, despite their autoreactive profile they are not deleted by central tolerance mechanisms, possibly because the level of autoreactivity is not registered as dangerous by deletion mechanisms or because editing of L-chain variable genes occurred which our experimental approach could not identify.

Physiological and Pathophysiological Roles of IgM Fc Receptor (FcµR) Isoforms

IgM is the first antibody to emerge during phylogeny, ontogeny, and immune responses and serves as a first line of defense. Effector proteins interacting with the Fc portion of IgM, such as complement and its receptors, have been extensively studied for their functions. IgM Fc receptor (FcµR), identified in 2009, is the newest member of the FcR family and is intriguingly expressed by lymphocytes only, suggesting the existence of distinct functions as compared to the FcRs for switched Ig isotypes, which are expressed by various immune and non-hematopoietic cells as central mediators of antibody-triggered responses by coupling the adaptive and innate immune responses. Results from FcµR-deficient mice suggest a regulatory function of FcµR in B cell tolerance, as evidenced by their propensity to produce autoantibodies of both IgM and IgG isotypes. In this article, we discuss conflicting views about the cellular distribution and potential functions of FcµR. The signaling function of the Ig-tail tyrosine-like motif in the FcµR cytoplasmic domain is now formally shown by substitutional experiments with the IgG2 B cell receptor. The potential adaptor protein associating with FcµR and the potential cleavage of its C-terminal cytoplasmic tail after IgM binding are still enigmatic. Critical amino acid residues in the Ig-like domain of FcµR for interacting with the IgM Cµ4 domain and the mode of interaction are now defined by crystallographic and cryo-electron microscopic analyses. Some discrepancies on these interactions are discussed. Finally, elevated levels of a soluble FcµR isoform in serum samples are described as the consequence of persistent B cell receptor stimulation, as seen in chronic lymphocytic leukemia and probably in antibody-mediated autoimmune disorders.

Upregulated Expression of the IL-9 Receptor on TRAF3-Deficient B Lymphocytes Confers Ig Isotype Switching Responsiveness to IL-9 in the Presence of Antigen Receptor Engagement and IL-4.

The pleiotropic cytokine IL-9 signals to target cells by binding to a heterodimeric receptor consisting of the unique subunit IL-9R and the common subunit γ-chain shared by multiple cytokines of the γ-chain family. In the current study, we found that the expression of IL-9R was strikingly upregulated in mouse naive follicular B cells genetically deficient in TNFR-associated factor 3 (TRAF3), a critical regulator of B cell survival and function. The highly upregulated IL-9R on Traf3-/- follicular B cells conferred responsiveness to IL-9, including IgM production and STAT3 phosphorylation. Interestingly, IL-9 significantly enhanced class switch recombination to IgG1 induced by BCR crosslinking plus IL-4 in Traf3-/- B cells, which was not observed in littermate control B cells. We further demonstrated that blocking the JAK-STAT3 signaling pathway abrogated the enhancing effect of IL-9 on class switch recombination to IgG1 induced by BCR crosslinking plus IL-4 in Traf3-/- B cells. Our study thus revealed, to our knowledge, a novel pathway that TRAF3 suppresses B cell activation and Ig isotype switching by inhibiting IL-9R-JAK-STAT3 signaling. Taken together, our findings provide (to our knowledge) new insights into the TRAF3-IL-9R axis in B cell function and have significant implications for the understanding and treatment of a variety of human diseases involving aberrant B cell activation such as autoimmune disorders.

Molecular and functional characterization of two IgM subclasses in large yellow croaker (Larimichthys crocea)

As the predominant immunoglobulin (Ig) isotype, IgM plays a crucial role in the acquired immunity of vertebrates. There is only one Igμ gene in mammals, except cattle, while the number of Igμ gene varies among teleost fish. In the current study, we found two functional Igμ genes (Igμ1 and Igμ2) and a pseudo Cμ gene (ψIgμ) in large yellow croaker (Larimichthys crocea). Both Igμ1 and Igμ2 genes possessed two transcript variants, which encoded the heavy chains of secreted (sIgM1 and sIgM2) and membrane-bound IgM1 and IgM2 (mIgM1 and mIgM2), respectively. Both the heavy chains of sIgM1 and sIgM2 consisted of a variable Ig domain, four constant Ig domains (CH1, CH2, CH3 and CH4) and a secretory tail, while those of mIgM1 and mIgM2 consisted of a variable Ig domain, three constant Ig domains (CH1, CH2 and CH3), a transmembrane domain and a short cytoplasmic tail. Cysteine residues that are necessary for the formation of intrachain and interchain disulfide bonds and tryptophan residues that are important for the folding of the Ig superfamily domain were well conserved in large yellow croaker IgM1 and IgM2. Interestingly, large yellow croaker IgM2 had an extra cysteine (C94) in the CH1 domain compared with IgM1, which may cause the structural difference between IgM1 and IgM2. A liquid chromatography–tandem mass spectrometry analysis revealed that both IgM1 and IgM2 were present at the protein level in large yellow croaker serum. Both the Igμ1 and Igμ2 genes were mainly expressed in systemic immune tissues, such as head kidney and spleen, but the expression level of Igμ2 was much lower than that of Igμ1. After Pseudomonas plecoglossicida infection, the expression levels of Igμ1 and Igμ2 in both the spleen and head kidney were significantly upregulated, with a higher upregulation of Igμ2 than that of Igμ1. These results suggested that Igμ1 and Igμ2 may play a differential role in the immune response of large yellow croaker against bacterial infection.

Levels of immunoglobulin isotypes in serum and respiratory samples of patients with chronic obstructive pulmonary disease: protocol for a systematic review and meta-analysis

IntroductionChronic obstructive pulmonary disease (COPD) is an inflammatory respiratory disorder characterised by the progressive worsening of lung function. Acute exacerbation of COPD (AECOPD) is a leading contributor to patient morbidity,...

From Castleman disease histopathological features to idiopathic multicentric Castleman disease: a multiparametric approach to exclude potential iMCD histopathological mimickers

AimsInternational consensus diagnostic criteria for idiopathic multicentric Castleman disease (iMCD) includes lymph node Castleman disease (CD) histopathological features as major criteria. Our aim was to apply those criteria in a...

Ataxia Telangiectasia Mutated and MSH2 Control Blunt DNA End Joining in Ig Class Switch Recombination.

Class-switch recombination (CSR) produces secondary Ig isotypes and requires activation-induced cytidine deaminase (AID)-dependent DNA deamination of intronic switch regions within the IgH (Igh) gene locus. Noncanonical repair of deaminated DNA by mismatch repair (MMR) or base excision repair (BER) creates DNA breaks that permit recombination between distal switch regions. Ataxia telangiectasia mutated (ATM)-dependent phosphorylation of AID at serine 38 (pS38-AID) promotes its interaction with apurinic/apyrimidinic endonuclease 1 (APE1), a BER protein, suggesting that ATM regulates CSR through BER. However, pS38-AID may also function in MMR during CSR, although the mechanism remains unknown. To examine whether ATM modulates BER- and/or MMR-dependent CSR, Atm-/- mice were bred to mice deficient for the MMR gene mutS homolog 2 (Msh2). Surprisingly, the predicted Mendelian frequencies of Atm-/-Msh2-/- adult mice were not obtained. To generate ATM and MSH2-deficient B cells, Atm was conditionally deleted on an Msh2-/- background using a floxed ATM allele (Atmf) and B cell-specific Cre recombinase expression (CD23-cre) to produce a deleted ATM allele (AtmD). As compared with AtmD/D and Msh2-/- mice and B cells, AtmD/DMsh2-/- mice and B cells display a reduced CSR phenotype. Interestingly, Sμ-Sγ1 junctions from AtmD/DMsh2-/- B cells that were induced to switch to IgG1 in vitro showed a significant loss of blunt end joins and an increase in insertions as compared with wild-type, AtmD/D, or Msh2-/- B cells. These data indicate that the absence of both ATM and MSH2 blocks nonhomologous end joining, leading to inefficient CSR. We propose a model whereby ATM and MSH2 function cooperatively to regulate end joining during CSR through pS38-AID.

Endogenously Synthesized n-3 fatty Acids in fat-1 Transgenic Mice Suppresses B Cell Activation

n-3 Polyunsaturated fatty acids (PUFAs) have been suggested as a preventive or therapeutic strategy for autoimmune or inflammatory diseases via the modulation of the activation, differentiation, and effector functions of various immune cells, especially T cells and macrophages. Despite the importance of B cells in the pathogenesis of autoimmune diseases, few studies have demonstrated their effect on B cells. In this study, we investigated the possible effects of n-3 PUFAs on B cell activation, differentiation, and isotype switching in fat-1 transgenic mice, which can convert n-6 PUFAs to n-3 PUFAs via n-3 desaturase derived from Caenorhabditis elegans. Fat-1 mice were fed a corn oil-rich diet for at least 4 weeks to increase the probability of converting n-6 PUFAs to n-3 PUFAs. Splenic B cells were then isolated, followed by stimulation with Toll-like receptor 4 or B cell receptor (BCR). Our results showed that increased n-3 PUFA levels in B cells by fat-1 overexpression inhibited B cell proliferation, cytokine secretion, such as interleukin (IL)-6, IL-10, tumor necrosis factor-α, plasma cell differentiation, and Ig isotype switching/secretion. These effects are possibly due to the suppression of Syk, Src, and AKT activation in B cells from fat-1 mice, as well as NF-κB. Collectively, our results suggest that fat-1 expression attenuates B cell responses, and n-3 PUFA supplementation can be a possible strategy to treat pathological conditions in which B cells play a key role.

The synergistic effects of the constant region and variable heavy chain families of multimeric immunoglobulin M on its interaction with Fc-mu receptor and antigen

Aim: As the primary response antibody with increasing use as a therapeutic immunoglobulin (Ig) format, IgM is also the largest antibody structure among the five major human isotypes. Spontaneously formed pentamers and hexamers of IgM have avidity effects that could compensate for weaker interactions in monomeric Igs. However, this advantage is counterbalanced by potential steric clashes when binding to multiple large antigens. Recent findings have challenged the expected canonical independence of Fc receptor (FcR) binding at the heavy chain constant (C)-region where the heavy chain C-region isotypes affected antigen binding at the variable (V)-regions, and the variable heavy (VH) families of the V-region affected FcR engagement at the antibody C-regions. With such effects found on other Ig isotypes, IgM candidates need to be investigated with regards to such effects, especially when considering its natural oligomerisation at the C-region that can amplify or modulate such allosteric effects. Methods: Through a panel of 14 recombinant complementarity determining regions (CDRs)-grafted trastuzumab and pertuzumab VH1-7 IgMs subjected to bio-layer interferometry measurements, the interactions with the antigen human epidermal growth factor receptor 2 (Her2), Fc-mu receptor (FcµR), and superantigen Protein L (PpL) were investigated. Results: Significant effects from the V-regions to mitigate FcµR binding and the IgM C-region bidirectional effect modulating Her2 antigen engagements at the V-regions were found. Additional modulatory effects from superantigen PpL binding on the V-region of the kappa chain (Vκ) mitigating antigen binding were also found, revealing possible novel mechanisms of antibody superantigens that can be moderated by the antibody VH frameworks.

XLID syndrome gene Med12 promotes Ig isotype switching through chromatin modification and enhancer RNA regulation.

The transcriptional coactivator Med12 regulates gene expression through its kinase module. Here, we show a kinase module-independent function of Med12 in CSR. Med12 is essential for super-enhancer activation by collaborating with p300-Jmjd6/Carm1 coactivator complexes. Med12 loss decreases H3K27 acetylation and eRNA transcription with concomitant impairment of AID-induced DNA breaks, S-S synapse formation, and 3'RR-Eμ interaction. CRISPR-dCas9-mediated enhancer activation reestablishes the epigenomic and transcriptional hallmarks of the super-enhancer and fully restores the Med12 depletion defects. Moreover, 3'RR-derived eRNAs are critical for promoting S region epigenetic regulation, synapse formation, and recruitment of Med12 and AID to the IgH locus. We find that XLID syndrome-associated Med12 mutations are defective in both 3'RR eRNA transcription and CSR, suggesting that B and neuronal cells may have cell-specific super-enhancer dysfunctions. We conclude that Med12 is essential for IgH 3'RR activation/eRNA transcription and plays a central role in AID-induced antibody gene diversification and genomic instability in B cells.

A Brazilian Real-Life Experience of Multiple Myeloma Patients: Final Results from the Mmybrave Multi-Center Study

Background: Despite incremental therapeutic improvements for patients (pt) with multiple myeloma (MM) over the past two decades, large discrepancies still exist worldwide in outcomes and in terms of access to novel agents and stem-cell transplantation (SCT). Moreover, the real-world effectiveness of novel therapies may not reflect pt profiles and outcomes from clinical trials. Management of MM in Brazil is subject to constraints that may impact clinical outcomes, and continual assessment of trends in treatment patterns and results in real life is warranted. Methods: The objective of MMyBRAve (NCT03506386) was to investigate the demographic and clinical characteristics, as well as the patterns of care and treatment results, for patients with MM treated at selected experienced hematology/oncology centers in Brazil. Eligible pts were aged ≥18 years and had been diagnosed with MM using contemporaneous IMWG criteria between 01/2008 and 12/2016. Demographic and clinical data were collected from institutional charts retrospectively between 06/2018 and 08/2019 using a dedicated electronic case report form. Treatment and patient evaluation were left to local standards of care. Baseline pt and disease characteristics, as well as treatment patterns, were analyzed descriptively. The overall survival (OS) was analyzed using the Kaplan-Meier method, and predictors of OS were explored using Cox proportional-hazards model. The OS was compared between subgroups defined according to selected baseline characteristics, eligibility to transplantation (Tx) at baseline, and type of institution (public vs private). Results: 17 institutions (Inst) contributed data from 943 pts, who had a mean age of 68.4 years (range, 29 to 103). Females comprised 46.3% of the pts, and 61.1% were reportedly White. Considering the 792 pts with information on immunoglobulin (Ig) isotype, 57.7% had IgG, 21.2% IgA, 16.9% light-chain only, non-secretory in 3.5%, and IgD, IgE or IgM in 0.6%. International Staging System (ISS) stage at diagnosis was I in 19.3%, II in 21.3%, III in 26.6%, and unknown in 32.8%. 491 (52.1%, 95% confidence interval [CI], 48.9% to 55.3%) pts were considered Tx-ineligible at baseline. 452 (47.9%, 95% CI, 44.7% to 51.1%) pts were considered Tx-eligible, and 250 (55.3% of 452; 95% CI, 50.6% to 60.0%) underwent Tx as part of frontline therapy. Overall, after a median follow-up of 63 months, the median OS was 70 months, with OS probabilities of 87% at 12 months, 78% at 24, 70% at 36, 62% at 48, and 55% at 60 months. Tx-ineligible pts were significantly older and had significantly higher levels of creatinine and lactic dehydrogenase (LDH), and more advanced ISS than Tx-eligible pts. Median OS times were 49 and 93 months for Tx-ineligible and Tx-eligible patients, with a hazard ratio (HR) of 0.52 favoring Tx-eligible (95% CI, 0.43 to 0.63; P<0.001). 519 pts came from public Inst, and 424 from private. The former had significantly lower levels of hemoglobin and significantly higher levels of creatinine, calcium and LDH, and more advanced ISS than pts from private Inst. Median OS in these two groups were 61 and 74 months, with a HR of 0.78 favoring pts from private Inst (95% CI, 0.64 to 0.94; P=0.010). Pts from public Inst were more likely to receive melphalan- or thalidomide-based frontline regimens, whereas those from private Inst were more likely to receive bortezomib in frontline. Other univariable baseline factors associated with OS were age, hemoglobin, creatinine, calcium, and Durie-Salmon stage. In multivariable analysis for factors associated with OS, all these remained significant, and so was Inst (HR=0.61, P=0.002). Although there was no significant difference in Tx eligibility between public and private Inst (46.1% vs 50.2%; P=0.224) or in the percentage of patients undergoing Tx (24.5% vs 29.0%; P=0.134), adding this variable to the multivariable model for OS suggested that it confounds the association between type of Inst and OS, since Tx eligibility became a stronger prognostic factor (HR=0.586, P<0.001) than type of Inst (HR=0.81, P=0.073). Conclusions: Comparing these results with those from previous studies, the OS of pts with MM is improving in Brazil. Discrepancies remain in OS for patients from public and private institutions. Although the reasons for this finding remain to be ascertained, differential access to novel therapies cannot be ruled out as a determining factor.

Anti-PF4 antibodies associated with disease severity in COVID-19

Severe COVID-19 is characterized by a prothrombotic state associated with thrombocytopenia, with microvascular thrombosis being almost invariably present in the lung and other organs at postmortem examination. We evaluated the presence of antibodies to platelet factor 4 (PF4)-polyanion complexes using a clinically validated immunoassay in 100 hospitalized patients with COVID-19 with moderate or severe disease (World Health Organization score, 4 to 10), 25 patients with acute COVID-19 visiting the emergency department, and 65 convalescent individuals. Anti-PF4 antibodies were detected in 95 of 100 hospitalized patients with COVID-19 (95.0%) irrespective of prior heparin treatment, with a mean optical density value of 0.871 ± 0.405 SD (range, 0.177 to 2.706). In contrast, patients hospitalized for severe acute respiratory disease unrelated to COVID-19 had markedly lower levels of the antibodies. In a high proportion of patients with COVID-19, levels of all three immunoglobulin (Ig) isotypes tested (IgG, IgM, and IgA) were simultaneously elevated. Antibody levels were higher in male than in female patients and higher in African Americans and Hispanics than in White patients. Anti-PF4 antibody levels were correlated with the maximum disease severity score and with significant reductions in circulating platelet counts during hospitalization. In individuals convalescent from COVID-19, the antibody levels returned to near-normal values. Sera from patients with COVID-19 induced higher levels of platelet activation than did sera from healthy blood donors, but the results were not correlated with the levels of anti-PF4 antibodies. These results demonstrate that the vast majority of patients with severe COVID-19 develop anti-PF4 antibodies, which may play a role in the clinical complications of COVID-19.

Prevention and Therapy of Metastatic HER-2+ Mammary Carcinoma with a Human Candidate HER-2 Virus-like Particle Vaccine.

Vaccines are a promising therapeutic alternative to monoclonal antibodies against HER-2+ breast cancer. We present the preclinical activity of an ES2B-C001, a VLP-based vaccine being developed for human breast cancer therapy. FVB mice challenged with HER-2+ mammary carcinoma cells QD developed progressive tumors, whereas all mice vaccinated with ES2B-C001+Montanide ISA 51, and 70% of mice vaccinated without adjuvant, remained tumor-free. ES2B-C001 completely inhibited lung metastases in mice challenged intravenously. HER-2 transgenic Delta16 mice developed mammary carcinomas by 4–8 months of age; two administrations of ES2B-C001+Montanide prevented tumor onset for >1 year. Young Delta16 mice challenged intravenously with QD cells developed a mean of 68 lung nodules in 13 weeks, whereas all mice vaccinated with ES2B-C001+Montanide, and 73% of mice vaccinated without adjuvant, remained metastasis-free. ES2B-C001 in adjuvant elicited strong anti-HER-2 antibody responses comprising all Ig isotypes; titers ranging from 1–10 mg/mL persisted for many months. Antibodies inhibited the 3D growth of human HER-2+ trastuzumab-sensitive and -resistant breast cancer cells. Vaccination did not induce cytokine storms; however, it increased the ELISpot frequency of IFN-γ secreting HER-2-specific splenocytes. ES2B-C001 is a promising candidate vaccine for the therapy of tumors expressing HER-2. Preclinical results warrant further development towards human clinical studies.

Enrichment of serum IgG4 in MuSK myasthenia gravis patients

Muscle-specific kinase (MuSK) myasthenia gravis (MG) is a neuromuscular autoimmune disease belonging to a growing group of IgG4 autoimmune diseases (IgG4-AIDs), in which the majority of pathogenic autoantibodies are of the IgG4 subclass. The more prevalent form of MG with acetylcholine receptor (AChR) antibodies is caused by IgG1–3 autoantibodies. A dominant role for IgG4 in autoimmune disease is intriguing due to its anti-inflammatory characteristics. It is unclear why MuSK autoantibodies are predominantly IgG4. We hypothesized that MuSK MG patients have a general predisposition to generate IgG4 responses, therefore resulting in high levels of circulating IgG4. To investigate this, we quantified serum Ig isotypes and IgG subclasses using nephelometric and turbidimetric assays in MuSK MG and AChR MG patients not under influence of immunosuppressive treatment. Absolute serum IgG1 was increased in both MuSK and AChR MG patients compared to healthy donors. In addition, only MuSK MG patients on average had significantly increased and enriched serum IgG4. Although more MuSK MG patients had elevated serum IgG4, for most the IgG4 serum levels fell within the normal range. Correlation analyses suggest MuSK-specific antibodies do not solely explain the variation in IgG4 levels. In conclusion, although serum IgG4 levels are slightly increased, the levels do not support ubiquitous IgG4 responses in MuSK MG patients as the underlying cause of dominant IgG4 MuSK antibodies.

Impact of Mycobacterium tuberculosis Infection on Human B Cell Compartment and Antibody Responses.

Tuberculosis (TB) remains one of the most important health challenges worldwide. Control of the TB epidemic has not yet been achieved because of the lack of an effective vaccine and rapid and sensitive diagnostic approaches, as well as the emergence of drug-resistant forms of M. tuberculosis. Cellular immunity has a pivotal role against M. tuberculosis infection, but the role of humoral immunity is still controversial. We analyzed the frequency, absolute counts, and phenotypic and functional subsets of B lymphocytes in the peripheral blood of patients with active TB and subjects with latent infection compared to healthy donors. Moreover, we analyzed serum levels of total Ig and their IgA, IgM, and IgG isotypes and the titers of preexisting antibodies against a pool of common viral pathogens. FlowCT and unsupervised clusterization analysis show that patients with active TB and LTBI subjects have modest non-significant reduction in the numbers of circulating B lymphocytes as compared to healthy donors. Moreover, LTBI subjects had high percentages of atypical B cell population and lower percentages of naive and switched memory B cells. These findings were supported by gene expression and GSEA analysis. Moreover, there were no differences between active TB patients, LTBI subjects and HD, either in serum levels of total Ig isotypes or in preexisting IgG antibody titers, to ten different antigens from eight common pathogenic viruses, clearly demonstrating that either active or latent M. tuberculosis infection preserves the antibody production capacity of long-lived plasma cells. Thus, our results agree with previous studies reporting unaltered B cell frequencies in the blood of active TB patients and LTBI individuals as compared to healthy controls.

Germline IgM predicts T cell immunity to Pneumocystis.

Pneumocystis is the most common fungal pulmonary infection in children under the age of 5 years. In children with primary immunodeficiency, Pneumocystis often presents at 3–6 months of age, a time period that coincides with the nadir of maternal IgG and when IgM is the dominant Ig isotype. Because B cells are the dominant antigen-presenting cells for Pneumocystis, we hypothesized the presence of fungal-specific IgMs in humans and mice and that these IgM specificities would predict T cell antigens. We detected fungal-specific IgMs in human and mouse sera and utilized immunoprecipitation to determine whether any antigens were similar across donors. We then assessed T cell responses to these antigens and found anti-Pneumocystis IgM in WT mice, Aicda–/– mice, and in human cord blood. Immunoprecipitation of Pneumocystis murina with human cord blood identified shared antigens among these donors. Using class II MHC binding prediction, we designed peptides with these antigens and identified robust peptide-specific lung T cell responses after P. murina infection. After mice were immunized with 2 of the antigens, adoptive transfer of vaccine-elicited CD4+ T cells showed effector activity, suggesting that these antigens contain protective Pneumocystis epitopes. These data support the notion that germline-encoded IgM B cell receptors are critical in antigen presentation and T cell priming in early Pneumocystis infection.

Altered Immunoglobulin Repertoire and Decreased IgA Somatic Hypermutation in the Gut during Chronic HIV-1 Infection.

ABSTRACTHumoral immune perturbations contribute to pathogenic outcomes in persons with HIV-1 infection (PWH). Gut barrier dysfunction in PWH is associated with microbial translocation and alterations in microbial communities (dysbiosis), and IgA, the most abundant immunoglobulin (Ig) isotype in the gut, is involved in gut homeostasis by interacting with the microbiome. We determined the impact of HIV-1 infection on the antibody repertoire in the gastrointestinal tract by comparing Ig gene utilization and somatic hypermutation (SHM) in colon biopsies from PWH (n = 19) versus age and sex-matched controls (n = 13). We correlated these Ig parameters with clinical, immunological, microbiome and virological data. Gene signatures of enhanced B cell activation were accompanied by skewed frequencies of multiple Ig Variable genes in PWH. PWH showed decreased frequencies of SHM in IgA and possibly IgG, with a substantial loss of highly mutated IgA sequences. The decline in IgA SHM in PWH correlated with gut CD4+ T cell loss and inversely correlated with mucosal inflammation and microbial translocation. Diminished gut IgA SHM in PWH was driven by transversion mutations at A or T deoxynucleotides, suggesting a defect not at the AID/APOBEC3 deamination step but at later stages of IgA SHM. These results expand our understanding of humoral immune perturbations in PWH that could have important implications in understanding mucosal immune defects in individuals with chronic HIV-1 infection.IMPORTANCE The gut is a major site of early HIV-1 replication and pathogenesis. Extensive CD4+ T cell depletion in this compartment results in a compromised epithelial barrier that facilitates the translocation of microbes into the underlying lamina propria and systemic circulation, resulting in chronic immune activation. To date, the consequences of microbial translocation on the mucosal humoral immune response (or vice versa) remains poorly integrated into the panoply of mucosal immune defects in PWH. We utilized next-generation sequencing approaches to profile the Ab repertoire and ascertain frequencies of somatic hypermutation in colon biopsies from antiretroviral therapy-naive PWH versus controls. Our findings identify perturbations in the Ab repertoire of PWH that could contribute to development or maintenance of dysbiosis. Moreover, IgA mutations significantly decreased in PWH and this was associated with adverse clinical outcomes. These data may provide insight into the mechanisms underlying impaired Ab-dependent gut homeostasis during chronic HIV-1 infection.