Pathogenic Abs Research Articles

Specific and Polyfunctional T Cell Response Against N-Methyl-d-aspartate Receptor in an Autoantibody-Mediated Encephalitis Model

Background: Anti-N-Methyl-d-aspartate receptor (NMDAR) autoimmune encephalitis (NMDAR AE) is an autoimmune disease characterized by severe psychiatric and neurological symptoms. While the pathogenic role of antibodies (Abs) directed against the GluN1 subunit of NMDAR is well described in this disease, the immune mechanisms involved in the generation of the autoimmune B cell response, especially the role of T helper cells, are poorly understood. Previously, we developed a B-cell-mediated mouse model of NMDAR AE by immunization with a GluN1359–378 peptide that drives a series of symptoms that recapitulate AE such as anxiety behaviour and spatial memory impairment. Results: In this mouse model, we identified anti-GluN1-specific CD4+ but also CD8+ T cells in both spleen and meninges. T helper cells have a polyfunctional profile, arguing for a T and B cell crosstalk to generate anti-GluN1 pathogenic Abs. Interestingly, proteomic analysis of AE meninges showed enrichment of differentially expressed proteins in biological processes associated with B cell activation and cytokine signalling pathways. Conclusions: This study identified, for the first time, a potential contribution of T helper cells in the pathology of NMDAR AE and paved the way for the development of future tolerogenic approaches to treat relapses.

HDAC6 Deletion Decreases Pristane-induced Inflammation.

Systemic lupus erythematosus is an autoimmune disease characterized by excessive inflammation and production of pathogenic Abs. Histone deacetylase 6 (HDAC6) is a class IIb histone deacetylase. It has been reported that selective HDAC6 inhibition decreases inflammation in lupus mouse models. In this study, sex- and age-matched wild-type (WT) and HDAC6-/- mice on the C57BL/6 background were administered 0.5 ml of pristane or PBS i.p. at 8-12 wk of age and were euthanized 10 d later. At sacrifice, body weight and spleen weight were measured, sera were collected, and splenocytes and peritoneal cells were harvested for flow cytometry. We found pristane administration increased the spleen weight with no difference between WT and HDAC6-/- mice. Pristane administration promoted the population of CD11b+Ly6C++ inflammatory monocytes and CD11b+Ly6G+ neutrophils. Peritoneal recruitment of these inflammatory monocytes and neutrophils was significantly decreased in HDAC6-/- mice compared with the WT mice. Flow cytometry results showed that the number of CD69+ T and B cells was increased in HDAC6-/- mice. Pristane administration also induced the IFN signature genes as determined by RT-qPCR. Furthermore, IFN signature genes were not affected in HDAC6-/- mice compared with the WT mice. In vitro studies in J774A.1 cells revealed that the selective HDAC6 inhibitor (ACY-738) increased acetylation of NF-κB while increasing Stat1 phosphorylation, which resulted in inducible NO synthase production in LPS/IFN-γ-stimulated cells. Taken together, these results demonstrate that although HDAC6 inhibition may inhibit some inflammatory pathways, others remain unaffected.

Concordance between radioimmunoassay and fixed cell-based assay in subjects without myasthenia gravis: optimizing the diagnostic approach.

Acetylcholine receptor antibody (AChR-Ab) detection is crucial in myasthenia gravis (MG) diagnosis and, currently, the radioimmunoassay (RIA) is the gold standard. However, RIA may detect AChR-Ab against nonpathogenic intracellular epitopes. In this study, we performed fixed cell-based assay (F-CBA) in RIA-AChR-Ab positive subjects without MG symptoms, to assess whether F-CBA could show a higher specificity compared to RIA in detecting pathogenic Abs. We reviewed medical records of patients referred to our MG outpatient clinic because of RIA-AChR-Ab detection. MG diagnosis was based on clinical examination, electrophysiology and Ab detection. AChR-Abs were tested by RIA in the whole cohort. Serum samples from RIA-positive asymptomatic subjects were retested by F-CBA. Of 605 subjects who tested RIA-AChR-Ab positive, MG diagnosis was confirmed in 599. Six subjects were RIA-AChR-Ab positive although they had never had MG symptoms; in four of these subjects AChR-Abs were not detected by F-CBA, whereas the remaining two (both non-MG thymoma cases) were positive also by F-CBA. RIA false positivity for AChR-Ab is very rare. Previous literature has demonstrated that F-CBA has higher sensitivity than RIA for MG, especially in ocular cases. Our preliminary results show that, in rare instances, F-CBA may be more specific than RIA for MG diagnosis.

Dissecting signaling parameters in autoreactive B cells

Abstract In systemic lupus erythematosus (SLE), IgG antibodies (Abs) directed against nuclear antigens (ANA) are pathogenic. Healthy individuals (HC) harbor a similar frequency of ANA+ mature B cells without producing pathogenic IgG Abs. Differences in behavior between ANA+/- B cells in SLE patients and HC have not been explored. The aim of this study was to measure the response to BCR activation in the ANA+/- B cell subpopulations in SLE patients and HC. B cells from 7 SLE patients and 5 HC were isolated and activated with F(ab)'2 fragments of anti-IgG Abs. Calcium flux was measured using indo-1 AM. The response was quantified by area under the curve (AUC), using the bound/unbound ratio of the marker for 5 minutes after activation. Values were compared using Wilcoxon rank sum test. In SLE patients, ANA+ IgG memory cells have a stronger calcium influx than ANA- (AUC for ANA + vs -: 89.46±18.51 vs 76.17±12.65; p=0.05). This difference is not present in HC (AUC for ANA + vs -: 88.02±16.51 vs 89.52±14.49; p=ns). In both HC and SLE patients, ANA+ naïve B cells are less responsive to BCR activation, suggesting anergy. IgM+ B cells overall, irrespective of ANA status, seem to be less responsive in patients with SLE. These results show that ANA+ IgG memory B cells are hyperreactive in SLE patients. The data also suggests a mechanism for the breach in tolerance that is observed in SLE. Further studies are warranted to explore the mechanism and effects on autoantibody generation in SLE.

Complement receptor 2 as a novel therapeutic target to suppress germinal center-derived autoantibody production in a SLE mouse model

Abstract Presence of spontaneous germinal center (GCs) in SLE patients correlates with the production of pathogenic, isotype-switched antibodies (Abs) and epitope spreading. In lupus mice, spontaneous GCs are a major site for self-reactive B cells to undergo clonal selection to self-antigens. Limited efficacy of current B cell-based lupus therapies highlights the need to explore alternative targets for inhibiting auto-Ab secreting B cells (ASCs) generation. We propose that complement receptor 2 (CD21) is a novel therapeutic target to block expansion of autoimmunity and pathogenic Ab production. To test this, we set-up mix bone marrow chimeras and adoptive transfer models with 564Igi lupus mice and human CD21-transgenic mice, which allowed us to selectively block CD21 function of either B cells or FDC, or both. CD21 blockade on B cells not only restricted recently activated B cells but also memory B cells (MBC) to participate in GC-derived ASC generation. CD21 blockade on FDCs intriguingly biased MBC fate towards ASC generation, though impeded recently activated B cell capacity to participate in GC response. Dual blockade of CD21 on B cells and FDC exhibited strongest inhibitory effect on GC-derived ASC generation and auto-Ab production. As expected, blockade of ASC generation by donor B cells further correlated with reduced Ab deposition and ameliorated kidney pathology. In brief, CD21 presents itself as a promising therapeutic target to suppress pathogenic auto-Ab generation in SLE.

Antiphospholipid Antibodies in Pregnancy: Maternal and Neonatal Implications.

Antiphospholipid antibodies (aPL Abs) have long been associated with the occurrence of certain specific pregnancy morbidities, affecting both mother and fetus. Antithrombotic-based prophylactic regimens are the standard of care. Their intensity is modulated by the thrombotic history and has greatly improved the prognosis related to spontaneous morbidity. Observational studies show that this treatment is still associated with the persistence of excess of late-pregnancy placental diseases, calling for new or complementary developments, yet to be validated. Rigorous prospective multicentric validation of clinical and laboratory parameters capable of identifying those women and fetuses at a risk of pejorative evolution, thus early prognosis, is a priority issue. These will make it possible to develop customized treatments and test them. Furthermore, there are still concerns, particularly neurodevelopmental ones, about children born to aPL Ab-positive mothers, and clarification based on regular, more systematic evaluations is required. Even after pregnancy, women with a pure obstetrical antiphospholipid syndrome are at a greater risk of venous and arterial thrombosis over time, and prevention needs to be improved. These women also appear to develop more psychiatric and mood disorders. Central nervous system imaging using high-resolution techniques has shown subtle impairments in the white matter, associated with the most pathogenic aPL Abs and the clinical significance of this is under investigation. These mothers also seem to develop an excess of cancers. The systemic impact of aPL Abs is gradually being suspected, although this requires further evidence, and prevention should be envisaged.

Naturally Occurring Anti-Idiotypic Antibodies Portray a Largely Private Repertoire in Immune-Mediated Thrombotic Thrombocytopenic Purpura.

Rare immune-mediated thrombotic thrombocytopenic purpura (iTTP) is a life-threatening disease resulting from a severe autoantibody-mediated ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 motifs, member 13) deficiency. Acute iTTP episodes are medical emergencies, but when treated appropriately >95% of patients survive. However, at least half of survivors will eventually experience a relapse. How remission of an initial episode is achieved and factors contributing to reemergence of anti-ADAMTS13 Abs and a relapsing course are poorly understood. In acquired hemophilia and systemic lupus erythematosus, anti-idiotypic Abs counteracting and neutralizing pathogenic autoantibodies contribute to remission. We selected and amplified the splenic anti-idiotypic IgG<sub>1</sub> Fab κ/λ repertoire of two relapsing iTTP patients on previously generated monoclonal inhibitory anti-ADAMTS13 Fabs by phage display to explore whether anti-idiotypic Abs have a role in iTTP. We obtained 27 single anti-idiotypic Fab clones, half of which had unique sequences, although both patients shared four H chain V region genes (V<sub>H</sub>1-69*01, V<sub>H</sub>3-15*01, V<sub>H</sub>3-23*01, and V<sub>H</sub>3-49*03). Anti-idiotypic Fab pools of both patients fully neutralized the inhibitor capacity of the monoclonal anti-ADAMTS13 Abs used for their selection. Preincubation of plasma samples of 22 unrelated iTTP patients stratified according to functional ADAMTS13 inhibitor titers (>2 Bethesda units/ml, or 1-2 Bethesda units/ml), with anti-idiotypic Fab pools neutralized functional ADAMTS13 inhibitors and restored ADAMTS13 activity in 18-45% of those cases. Taken together, we present evidence for the presence of an anti-idiotypic immune response in iTTP patients. The interindividual generalizability of this response is limited despite relatively uniform pathogenic anti-ADAMTS13 Abs recognizing a dominant epitope in the ADAMTS13 spacer domain.

Label-Free Detection and Characterization of Heparin-Induced Thrombocytopenia (HIT)-like Antibodies.

Heparin-induced thrombocytopenia (HIT) antibodies (Abs) can mediate and activate blood cells, forming blood clots. To detect HIT Abs, immunological assays with high sensitivity (≥95%) and fast response are widely used, but only about 50% of these tests are accurate as non-HIT Abs also bind to the same antigens. We aim to develop biosensor-based electrical detection to better differentiate HIT-like from non-HIT-like Abs. As a proof of principle, we tested with two types of commercially available monoclonal Abs including KKO (inducing HIT) and RTO (noninducing HIT). Platelet factor 4/Heparin antigens were immobilized on gold electrodes, and binding of antibodies on the chips was detected based on the change in the charge transfer resistance (Rct). Binding of KKO on sensors yielded a significantly lower charge transfer resistance than that of RTO. Bound antibodies and their binding characteristics on the sensors were confirmed and characterized by complementary techniques. Analysis of thermal kinetics showed that RTO bonds are more stable than those of KKO, whereas KKO exhibited a higher negative ζ potential than RTO. These different characteristics made it possible to electrically differentiate these two types of antibodies. Our study opens a new avenue for the development of sensors for better detection of pathogenic Abs in HIT patients.

Luminex Multiplex Bead Assay Monitoring HLA IgG Antibodies in Sensitized Pre- and Post-transplant Patients: Clonality of the Detection Antibody Impacts Specificity and Sensitivity

The number and the binding affinity, measured as the mean fluorescent intensity (MFI) of HLA-specific IgG antibodies, formed in the sera of end-stage organ disease patients and allograft recipients, referred to as sensitization, may restrict the availability of a donor organ and/or lead to graft failure after transplantation. The MFI of HLA Abs in sera is monitored with the Luminex-based single-antigen bead (SAB) immunoassay. The following two factors may impact the reliable measurement of MFI: one, the HLA structural variants on the SAB, namely, trimeric HLA (closed conformers, CC) and monomeric heavy chains (open conformers, OC); and two, the nature of the detection Abs, namely, IgG heavy-chain binding polyclonal-Fab (IgHPolyFab) or Fc-binding monoclonal-IgG (FcMonoIgG). Anti-CC Abs correlate with positive flow cross-matches, and are considered to be pathogenic and damaging to the graft, whereas anti-OC Abs appear to have little relevance to graft attrition. The presence of both CC and OC on beads may impair the reliability of monitoring the nature and MFI of pathogenic Abs. Our objective is to compare the MFI of the HLA Abs in the sera of 20 sensitized patients in two different SAB assays, with the two detection Abs. Our data reveal that the admixture of OC with CC on beads will affect the reliability of the measurement of the pathogenic Abs, and that FcMonoIgG is the more sensitive and specific detection Ab for the accurate assessment of HLA sensitization.

Fc-Receptor Targeted Therapies for the Treatment of Myasthenia gravis.

Myasthenia gravis (MG) is an autoimmune disease in which immunoglobulin G (IgG) antibodies (Abs) bind to acetylcholine receptors (AChR) or to functionally related molecules in the postsynaptic membrane at the neuromuscular junction. IgG crystallizable fragment (Fc)-mediated effector functions, such as antibody-dependent complement deposition, contribute to disease development and progression. Despite progress in understanding Ab-mediated disease mechanisms, immunotherapy of MG remained rather unspecific with corticosteroids and maintenance with immunosuppressants as first choice drugs for most patients. More specific therapeutic IgG Fc-based platforms that reduce serum half-life or effector functions of pathogenic MG-related Abs are currently being developed, tested in clinical trials or have recently been successfully translated into the clinic. In this review, we illustrate mechanisms of action and clinical efficacies of emerging Fc-mediated therapeutics such as neonatal Fc receptor (FcRn)-targeting agents. Furthermore, we evaluate prospects of therapies targeting classical Fc receptors that have shown promising therapeutic efficacy in other antibody-mediated conditions. Increased availability of Fc- and Fc receptor-targeting biologics might foster the development of personalized immunotherapies with the potential to induce sustained disease remission in patients with MG.

Allosensitization in Heart Transplantation: The Importance of a Comprehensive Approach

<h3>Introduction</h3> HLA sensitization presents a major barrier to transplantation yet sensitive techniques such as the Luminex Single Antigen Bead (SAB) assay, when used in isolation, may inappropriately limit the donor pool. We report a case of apparent robust HLA sensitization following HBV vaccination and describe our approach to the assessment of HLA reactivity leading to successful heart transplantation (HTx). <h3>Case Report</h3> A 50 year old Caucasian male with long standing ARVD developed recurrent VT and worsening RV dysfunction leading to HTx evaluation. There was no history of transfusion, surgery, or mechanical circulatory support. The Luminex SAB assay at listing and serially thereafter was negative. Unexpectedly, broad class II HLA sensitization (cPRA 98%) was suddenly detected by SAB assay after HBV vaccination concerning for either a heterologous immune response or false reactivity to the beads. Multiple antibodies (Abs) persisted at 1:8 dilution and included Abs against the recipients own HLA (autologous flow XM negative). At the time when a donor became available, 4 DSA were identified on the most recent Luminex SAB. Given his high cPRA in the absence of a strong allosensitizing event, we combined assessment with an alternative solid phase assay (LABScreen PRA), 4-digit donor typing, and prospective crossmatches by Flow Cytometry (FC) and Complement Dependent Cytotoxicity (CDC) to determine the safety of proceeding with HTx (Figure 1). Notably, no HLA Abs were detected using the LABScreen PRA and both FC and CDC crossmatches were negative. The patient underwent successful HTx with no rejection and normal graft function. <h3>Summary</h3> HLA allosensitization portends worse outcomes after transplant. However, the high sensitivity of the Luminex SAB and potential for false positivity may result in inappropriately limiting access to HTx. This case highlights the importance of comprehensive assessment of HLA sensitization in situations of uncertainty to help identify pathogenic HLA Abs and allow for successful HTx.

Memory CD8 T cell responses are impaired during Zika virus infection in mice

Abstract Nearly half the world’s population is at risk for infection by Zika virus (ZIKV), the causative agent of congenital Zika syndrome in infants and Guillain-Barre syndrome in adults. ZIKV vaccination strategies are currently focused on neutralizing antibody (Ab) generation, yet recent evidence suggests that Abs may contribute to pathogenesis under certain conditions. In contrast, increasing evidence supports a key role for T cells in protecting against ZIKV. We have begun to evaluate the generation of memory T cell responses to ZIKV infection in unmanipulated immunocompetent mice. Four-week-old wild-type C57BL/6 mice were infected with ZIKV for 30 days, followed by challenge with ZIKV and evaluation of memory CD4 and CD8 T cell responses at day 3 after secondary ZIKV infection. As expected, total numbers of CD4 and CD8 T cells expanded during secondary ZIKV challenge when compared to primary ZIKV responses at 7 days post-infection. However, only antigen-experienced CD4 T cells (CD11a+CD49d+) and not antigen-experienced CD8 T cells (CD11a+CD8lo) were similarly expanded in secondary infection. These CD8 T cell responses were comprised of short-lived effector cells and not memory precursors, indicating that CD8 T cells memory responses are impaired after primary ZIKV infection. These data suggest potential mechanisms of immune-evasion by ZIKV, and ongoing studies are focused on defining the mechanisms by which ZIKV inhibits the generation of memory CD8 T cell responses. Our findings should offer new strategies for developing ZIKV vaccines that enhance protective T cell responses and avoid pathogenic Ab responses.

Antibody Cloning Identifies Pathogenic and Non-Pathogenic Antibodies in Heparin-Induced Thrombocytopenia and Defines the Molecular Signatures That Differentiate the Two Types of Antibodies

Heparin-induced thrombocytopenia (HIT) is a common adverse drug reaction associated with frequent life-threatening thrombotic complications. The hallmark of HIT is polyclonal antibodies (Abs) that recognize platelet alpha granule chemokine PF4 when it binds to heparin (PF4/H). These Abs can be detected in solid phase assays that use PF4/H as a target (PF4 ELISA), but only a minority of patients testing positive actually have HIT, i.e., most heparin-induced Abs are non-pathogenic. In patients who have clinical HIT, Abs that activate platelets can be detected using a platelet-activation assay such as the serotonin release assay, or the PF4-dependent p-selectin expression assay (PEA) (Chest 2016; 150:506). Thus, there are at least two distinct types of heparin-induced Abs - those that react only in PF4 ELISA and are seemingly "non-platelet-activating" and "non-pathogenic" and those that are "platelet-activating" and "pathogenic". To date, the molecular basis for the differing clinical and serologic behaviors of pathogenic and non-pathogenic Abs is uncertain. To address this issue, we performed single cell cloning to clone B cell receptors from IgG1+ B cells from HIT patients. We deposited single B cells (CD19+IgG1+) from 6 patients with "classical" and 2 patients with "spontaneous" HIT into 96 well plates containing feeder cells (from G Kelsoe, Duke U) that support B cell proliferation and Ab secretion (Immunity 2018;48:174). Clones secreting IgG were first screened in PF4 ELISA and positive results were obtained with 55 clones from 6 patients. Further screening showed that 7 of these clones (from 4 patients) were also PEA-positive (platelet-activating). Clones positive only in PF4 ELISA, positive in both PF4 ELISA and PEA, or negative in PF4 ELISA were designated NP (non-pathogenic), PA (platelet-activating) and NB (non-binding), respectively. H and L chain variable regions were defined in 7 PA, 42 NP and 34 NB clones. The following findings were made when sequences in the 3 clonal groups were compared: PA clones preferentially used JH6 (p=0.002) and the VH3/JH6 combination (p=0.0003)The PA and NP Abs all employed κ chains, whereas κ chain usage for NB clones was 61% (p&amp;lt;0.0001).No preferred signatures were identified in κ chain complementarity determining regions (LCDR3) of PA clones that differentiate them from NP and NB Abs.PA Abs had longer heavy chain CDR3s (HCDR3) than NP (p&amp;lt;0.001) or NB (p=0.0001) AbsPA Abs contained more positively charged amino acid residues compared to NP (p=0.058) or NB (p=0.002) Abs.PA Abs contained more tyrosine residues compared to NP (p=0.067) or NB (p&amp;lt;0.0001) AbsFive of 7 PA clones contained an RX1-2K/RX1-2R/H (RKH) motif in HCDR3; the remaining 2 PA clones contained a string of at least 5 tyrosines (Y5 motif) in HCDR3. The RKH and Y5 motifs were not found in any of the 76 NP and NB clones. Substitution of alanine for positively charged residues of the RKH motif or of tyrosine residues in the Y5 motif in PA clones reduced PF4/H binding and platelet activation, arguing for functional significance of both motifs. Utilization of nearly identical H and L chains within 3 groups of clones and of shared H chains within 3 groups of clones (both PA and NP) was observed in multiple patients. Moreover, utilization of a shared H chain was observed within 3 NP clones from two unrelated patients. These findings indicate clonal amplification and convergence of the B cell (both PA and NP) response, likely in response to a common antigen. High throughput sequencing of IgG H chains were performed on peripheral blood mononuclear cells (PBMC) from 7 HIT patients and 3 healthy donors. Eleven of 1585 H chain sequences (0.69%) from HIT patients contained the RKH and 18 (1.1%) contained the Y5 motif. In 3 healthy donors, 4 of 1418 H chain sequences (0.28%) contained RKH and none (0%) contained Y5. The findings reflect amplification of B cells with receptors containing RKH and Y5 motifs in HIT patients (p=0.1 for excess RKH and p&amp;lt;0.0001 for Y5 in HIT). These observations provide the first characterization of Ig structural motifs that are favored for selection in the humoral immune response leading to HIT and suggest that the RKH and Y5 CDR3 motifs in particular may contribute importantly to Ab pathogenicity. Findings made are expected to facilitate further work to define features specific to "pathogenic" HIT Abs and, possibly, to identify genetic variants that predispose individuals to experience HIT. Disclosures Padmanabhan: Terumo BCT: Consultancy; Veralox Therapeutics: Membership on an entity's Board of Directors or advisory committees; Versiti Wisconsin: Patents &amp; Royalties: Related to HIT patents; Retham Technologies: Equity Ownership; Janssen R&amp;D: Consultancy.

Frequency and Clinical Impact of Platelet Factor 4-Specific Antibodies in Patients Undergoing Extracorporeal Membrane Oxygenation.

Extracorporeal membrane oxygenation (ECMO) provides circulatory support in patients with severe heart failure, but the frequent use of unfractionated heparin exposes patients to high risk of heparin-induced thrombocytopenia (HIT). We prospectively evaluated the development and clinical impact of platelet factor 4 (PF4)-specific antibodies (Abs) during ECMO and whether specific biological characteristics could predict HIT. From 2014 to 2018, we studied 57 adults who underwent an ECMO for at least 5 days. The plasma samples collected daily were tested for PF4-specific Abs using immunoassays to detect immunoglobulin (Ig) G, A, and M isotypes or only IgG. Serotonin release assay was performed without and with PF4 to detect pathogenic Abs. Twenty-nine patients (50%) were positive for PF4-specific Abs (IgG, A, M), with IgG in 17/57 (30%) and 16 of them (94%) were immunized within 10 days. PF4-specific IgG Abs did not affect the clinical or biological course of most patients. HIT was suspected in only two patients with ECMO circuit dysfunction and unexpected platelet count decrease after day 5. High levels of PF4-specific IgG were detected in both patients, and HIT was confirmed by a serotonin release assay, which was also more sensitive when exogenous PF4 was present. PF4-specific Abs are common during ECMO but are mostly non-pathogenic and not associated with a less favorable prognosis. However, an abnormal platelet count evolution, in particular if associated with ECMO circuit dysfunction, should prompt the search for pathogenic PF4-specific IgG.

Nanoparticles Bearing TSH Receptor Protein and a Tolerogenic Molecule Do Not Induce Immune Tolerance but Exacerbate Thyroid Autoimmunity in hTSHR/NOD.H2h4 Mice.

Transgenic NOD.H2h4 mice that express the human (h) TSHR A-subunit in the thyroid gland spontaneously develop pathogenic TSHR autoantibodies resembling those in patients with Graves disease. Nanoparticles coupled to recombinant hTSHR A-subunit protein and a tolerogenic molecule (ligand for the endogenous aryl-hydrocarbon receptor; ITE) were injected i.p. four times at weekly intervals into hTSHR/NOD.H2h4 mice with the goal of blocking TSHR Ab development. Unexpectedly, in transgenic mice, injecting TSHR A-subunit-ITE nanoparticles (not ITE-nanoparticles or buffer) accelerated and enhanced the development of pathogenic TSHR Abs measured by inhibition of TSH binding to the TSHR. Nonpathogenic TSHR Abs (ELISA) were enhanced in transgenics and induced in wild-type littermates. Serendipitously, these findings have important implications for disease pathogenesis: development of Graves TSHR Abs is limited by the availability of A-subunit protein, which is shed from membrane bound TSHR, expressed at low levels in the thyroid. The enhanced TSHR Ab response following injected TSHR A-subunit protein-nanoparticles is reminiscent of the transient increase in pathogenic TSHR Abs following the release of thyroid autoantigens after radio-iodine therapy in Graves patients. However, in the hTSHR/NOD.H2h4 model, enhancement is specific for TSHR Abs, with Abs to thyroglobulin and thyroid peroxidase remaining unchanged. In conclusion, despite the inclusion of a tolerogenic molecule, injected nanoparticles coated with TSHR A-subunit protein enhanced and accelerated development of pathogenic TSHR Abs in hTSHR/NOD. NOD.H2h4 These findings emphasize the need for sufficient TSHR A-subunit protein to activate the immune system and the generation of stimulatory TSHR Abs in genetically predisposed individuals.

Distinct Binding Characteristics of Pathogenic Anti-Platelet Factor-4/Polyanion Antibodies to Antigens Coated on Different Substrates: A Perspective on Clinical Application.

The polyanion heparin, which is frequently used in patients, complexes with the platelet-derived cationic chemokine platelet factor (PF4, CXCL4). This results in the formation of anti-PF4/heparin antibodies (anti-PF4/H Abs). Anti-PF4/H Abs are classified into three groups: (i) nonpathogenic Abs (group 1) with no clinical relevance; (ii) pathogenic heparin-dependent Abs (group 2), which activate platelets and can cause the severe adverse drug effect heparin-induced thrombocytopenia (HIT); and (iii) pathogenic autoimmune-HIT Abs (group 3), in which group 3 anti-PF4/H Abs causes a HIT-like autoimmune disease in the absence of heparin. Enzyme immunoassays using PF4/H complexes coated on the solid phase for detection of anti-PF4/H Abs cannot differentiate between pathogenic and nonpathogenic anti-PF4/H Abs. By single-molecule force spectroscopy, we identify a specific feature of pathogenic group 2 and group 3 Abs antibodies that (in contrast to nonpathogenic group 1 Abs) their binding forces to PF4/H complexes coated on platelets were significantly higher compared with those of PF4/H complexes immobilized on a solid phase. Only group 3 Abs showed high binding forces to platelets without the addition of PF4. In the presence of 50 μg/mL PF4, group 2 Abs also showed high binding forces to platelets. In contrast, binding forces of group 1 Abs always remained low (<100 pN). Our findings may have major relevance for the development of clinically applicable solid-phase assays, which allow differentiation of pathogenic platelet-activating from nonpathogenic anti-PF4/H Abs. Membrane-based expression of antigens might also increase the specificity of other assays for the detection of pathogenic (auto)-antibodies in clinical medicine.

Frequency of Anti-PF4/Heparin Antibodies in Patients Treated with Extra-Corporeal Membrane Oxygenation and Detection By Sensitized Serotonin Release Assay of Pathogenic Heparin-Induced Thrombocytopenia Antibodies

Introduction: Extra-Corporeal Membrane Oxygenation (ECMO) provides circulatory support in case of severe cardiac failure and is associated, as cardiopulmonary bypass, with strong activation of platelets, which likely leads to the release in patient blood of large amounts of platelet factor 4 (PF4). Therefore, systematic use of unfractionated heparin (UFH) for maintaining patency of circuits and to prevent thrombosis, exposes patients to heparin-induced thrombocytopenia (HIT). However, the risk of HIT in this particular clinical setting remains poorly documented.Objectives: In this monocentric study, the frequency of development of antibodies to PF4 modified by heparin (anti-PF4/H Abs) was prospectively evaluated in patients who had underwent ECMO, and the impact of these Abs on platelet count (PC) and clinical course was assessed.Patients and Methods: From February 2014 to January 2018, we enrolled in the University Hospital of Tours all consecutive adult patients with ECMO and treated with UFH for at least 5 days. Plasma samples were collected daily, and PF4-specific Abs were systematically detected using HAT45® (GTI), an ELISA assaying IgG, IgA and IgM isotypes, and another kit specific for IgG to modified PF4 (HAT45G®; GTI). Serotonin release assay (SRA) was also performed to detect platelet-activating Abs (and therefore potentially pathogenic) in plasma samples containing significant levels of anti-PF4/H IgG. Noticeably, SRA was also done after adding 10 μg/ml of PF4 in the reaction mixture (PF4-SRA), since we recently showed this modification might improve the detection of pathogenic HIT antibodies (Vayne et al, Brit J Haematol, 179(5):811-819, 2017). Demographics, PC, and clinical events were collected in all patients until the end of ECMO.Results: Fifty-seven patients with a median age of 57 years (range: 24-76) and who were mainly men (sex ratio W/M: 0.3) were enrolled. ECMO was performed due to medical issues (dilated cardiomyopathy, myocardial infarction, acute myocarditis) in 70 % of cases and in surgical settings in the others. Significant titres of anti-PF4/H IgG/A/M Abs were detected before ECMO in 6 of 57 patients (10.5%), with anti-PF4/H IgG present in only 3 cases (5%). After ECMO was initiated, 28 patients (49%) developed IgG/A/M anti-PF4/H Abs, with anti-PF4/H IgG present in 17 of them (30%). Half of these patients were immunized within 5 days after ECMO initiation and 95% of them within 10 days, with a median delay of 8 days before Abs detection (range: 4-26 days). Only 3 patients (5%) exhibited pathogenic anti-PF4/H IgG with positive SRA and the level of platelet release was always stronger when the test was performed with exogenous PF4 (SRA-PF4). Moreover, in one patient, pathogenic HIT Abs were detected earlier by PF4-SRA, i.e. 2 days before conventional SRA was positive. An abnormal PC pattern was evidenced in 2 of the 3 patients with positive SRA, with a dramatic PC fall after day 5 (of 49% and 31.5 % between days 9-10 and 5-6 respectively). The diagnosis of HIT was confirmed in these 2 cases, which also had suggestive clinical manifestations (arterial thrombosis and cutaneous necrosis). In the third patient, HIT was not suspected, because of low PC before ECMO with persistent thrombocytopenia post-operatively and early death on day 9. In the patients with negative SRA, the development of anti-PF4/H IgG did not appear to impact the biological and clinical evolution, with similar rates of thrombosis and death than in non-immunized patients. Therefore, patients with non-pathogenic anti-PF4/H IgG displayed the same PC pattern than non-immunized patients, with decreasing PC from ECMO initiation until day 5, followed by a slow PC recovery.Conclusion: The development of anti-PF4/H Abs is frequent during ECMO but these antibodies are rarely pathogenic, and not associated with a less favourable prognosis. PC evolution analysis during ECMO appears reliable for suspecting HIT, and a dramatic PC fall or a non-recovery of PC after day 5 should prompt to look for platelet-activating anti-PF4/H Abs. Therefore, IgG specific immunoassays should always be combined with platelet activation tests to improve the specificity of HIT diagnosis in this particular clinical condition. Interestingly, the addition of exogenous PF4 when performing SRA could allow an earlier detection of HIT pathogenic antibodies in patients undergoing ECMO. DisclosuresNo relevant conflicts of interest to declare.

CD83 Antibody Inhibits Human B Cell Responses to Antigen as well as Dendritic Cell-Mediated CD4 T Cell Responses.

Anti-CD83 Ab capable of Ab-dependent cellular cytotoxicity can deplete activated CD83+ human dendritic cells, thereby inhibiting CD4 T cell-mediated acute graft-versus-host disease. As CD83 is also expressed on the surface of activated B lymphocytes, we hypothesized that anti-CD83 would also inhibit B cell responses to stimulation. We found that anti-CD83 inhibited total IgM and IgG production in vitro by allostimulated human PBMC. Also, Ag-specific Ab responses to immunization of SCID mice xenografted with human PBMC were inhibited by anti-CD83 treatment. This inhibition occurred without depletion of all human B cells because anti-CD83 lysed activated CD83+ B cells by Ab-dependent cellular cytotoxicity and spared resting (CD83-) B cells. In cultured human PBMC, anti-CD83 inhibited tetanus toxoid-stimulated B cell proliferation and concomitant dendritic cell-mediated CD4 T cell proliferation and expression of IFN-γ and IL-17A, with minimal losses of B cells (<20%). In contrast, the anti-CD20 mAb rituximab depleted >80% of B cells but had no effect on CD4 T cell proliferation and cytokine expression. By virtue of the ability of anti-CD83 to selectively deplete activated, but not resting, B cells and dendritic cells, with the latter reducing CD4 T cell responses, anti-CD83 may be clinically useful in autoimmunity and transplantation. Advantages might include inhibited expansion of autoantigen- or alloantigen-specific B cells and CD4 T cells, thus preventing further production of pathogenic Abs and inflammatory cytokines while preserving protective memory and regulatory cells.

Analysis of peripheral B cells and autoantibodies against the anti-nicotinic acetylcholine receptor derived from patients with myasthenia gravis using single-cell manipulation tools.

The majority of patients with myasthenia gravis (MG), an organ-specific autoimmune disease, harbor autoantibodies that attack the nicotinic acetylcholine receptor (nAChR-Abs) at the neuromuscular junction of skeletal muscles, resulting in muscle weakness. Single cell manipulation technologies coupled with genetic engineering are very powerful tools to examine T cell and B cell repertoires and the dynamics of adaptive immunity. These tools have been utilized to develop mAbs in parallel with hybridomas, phage display technologies and B-cell immortalization. By applying a single cell technology and novel high-throughput cell-based binding assays, we identified peripheral B cells that produce pathogenic nAChR-Abs in patients with MG. Although anti-nAChR antibodies produced by individual peripheral B cells generally exhibited low binding affinity for the α-subunit of the nAChR and great sequence diversity, a small fraction of these antibodies bound with high affinity to native-structured nAChRs on cell surfaces. B12L, one such Ab isolated here, competed with a rat Ab (mAb35) for binding to the human nAChR and thus considered to recognize the main immunogenic region (MIR). By evaluating the Ab in in vitro cell-based assays and an in vivo rat passive transfer model, B12L was found to act as a pathogenic Ab in rodents and presumably in humans.These findings suggest that B cells in peripheral blood may impact MG pathogenicity. Our methodology can be applied not only to validate pathogenic Abs as molecular target of MG treatment, but also to discover and analyze Ab production systems in other human diseases.

Accelerated Systemic Autoimmunity in the Absence of Somatic Hypermutation in 564Igi: A Mouse Model of Systemic Lupus with Knocked-In Heavy and Light Chain Genes.

564Igi mice have knocked-in immunoglobulin (Ig) heavy (H) and light (L) chain genes that encode an autoantibody recognizing RNA. Previously, we showed that these mice produce pathogenic IgG autoantibodies when activation-induced deaminase (AID) is expressed in pre-B and immature B cells but not when it is expressed only in mature B cells. AID has two functions; it is necessary for somatic hypermutation (SHM) and class switch recombination (CSR). To determine the role of each of these functions in the generation of pathogenic autoantibodies, we generated 564Igi mice that carry a mutant AID-encoding gene, Aicda (AicdaG23S), which is capable of promoting CSR but not SHM. We found that 564Igi AicdaG23S mice secreted class-switched antibodies (Abs) at levels approximately equal to 564Igi mice. However, compared to 564Igi mice, 564Igi AicdaG23S mice had increased pathogenic IgG Abs and severe systemic lupus erythematosus-like disease, including, glomerulonephritis, and early death. We suggest that in 564Igi mice SHM by AID changes Ig receptors away from self reactivity, thereby mitigating the production of autoantibody, providing a novel mechanism of tolerance.