IgG-Fc glycosylation before and after rituximab treatment in immune thrombocytopenia

The interactions of antibodies with myeloid Fc gamma receptors and the complement system are regulated by an Asn297-linked glycan in the Fc portion of IgG. Alterations of serum IgG-Fc glycosylation have been reported in various autoimmune diseases, and correlate with treatment response and disease activity. We hypothesized that IgG-Fc glycosylation is altered in immune thrombocytopenia (ITP) and associates with response to anti-CD20 monoclonal antibody treatment (rituximab). IgG-Fc glycosylation was analyzed by liquid chromatography-mass spectrometry. We found that IgG-Fc glycosylation was identical between refractory ITP patients (HOVON64 trial; N = 108) and healthy controls (N = 120). Two months after rituximab treatment, we observed a shift in Fc glycosylation, with a mean 1.7% reduction in galactosylation for IgG1 and IgG4 and a mean 1.5% increase for bisection in IgG1, IgG2/3 and IgG4 (adjusted p < 1.7 x 10(-3) and p < 2 x 10(-4), respectively). Neither baseline nor longitudinal changes in IgG-Fc glycosylation after rituximab were associated with clinical treatment response. We conclude that IgG-Fc glycosylation in refractory ITP is similar to healthy controls and does not predict treatment responses to rituximab. The observed changes two months after treatment suggest that rituximab may influence total serum IgG-Fc glycosylation. Overall, our study suggests that the pathophysiology of refractory ITP may differ from other autoimmune diseases.Proteomic

Clinical characteristics of human platelet antigen (HPA)-1a and HPA-5b alloimmunised pregnancies and the association between platelet HPA-5b antibodies and symptomatic fetal neonatal alloimmune thrombocytopenia

Fetal neonatal alloimmune thrombocytopenia (FNAIT) is caused by maternal alloantibodies directed against the human platelet antigens (mostly HPA-1a or HPA-5b) of the (unborn) child and can lead to severe bleeding. Anti-HPA-1a-mediated FNAIT shows a severe clinical outcome more often than anti-HPA-5b-mediated FNAIT. Given the relatively high prevalence of anti-HPA-5b in pregnant women, the detection of anti-HPA-5b in FNAIT-suspected cases may in some cases be an incidental finding. Therefore we investigated the frequency of anti-HPA-5b-associated severe bleeding in FNAIT. We performed a retrospective nationwide cohort study in cases with clinical suspicion of FNAIT. HPA antibody screening was performed using monoclonal antibody-specific immobilisation of platelet antigens. Parents and neonates were typed for the cognate antigen. Clinical data were collected by a structured questionnaire. In 1 864 suspected FNAIT cases, 161 cases (8 center dot 6%) had anti-HPA-1a and 60 (3 center dot 2%) had anti-HPA-5b. The proportion of cases with severe bleeding did not differ between the cases with anti-HPA-1a (14/129; 11%) and anti-HPA-5b (4/40; 10%). In multigravida pregnant women with a FNAIT-suspected child, 100% (81/81) of anti-HPA-1a cases and 79% (38/48) of anti-HPA-5b cases were HPA-incompatible, whereas 86% and 52% respectively were expected, based on the HPA allele distribution. We conclude that anti-HPA-5b can be associated with severe neonatal bleeding symptoms. A prospective study is needed for true assessment of the natural history of anti-HPA-5b mediated FNAIT.Developmen

Biological stratification of clinical disease courses in childhood immune thrombocytopenia

Background In childhood immune thrombocytopenia (ITP), an autoimmune bleeding disorder, there is a need for better prediction of individual disease courses and treatment outcomes.Objective To predict the response to intravenous immunoglobulins (IVIg) and ITP disease course using genetic and immune markers.Methods Children aged younger than 7 years with newly diagnosed ITP (N = 147) from the Treatment With or Without IVIG for Kids with ITP study were included, which randomized children to an IVIg or observation group. A total of 46 variables were available: clinical characteristics, targeted genotyping, lymphocyte immune phenotyping, and platelet autoantibodies.Results In the treatment arm, 48/80 children (60%) showed a complete response (platelets >= 100 x 10(9)/L) that lasted for at least 1 month (complete sustained response [CSR]) and 32 exhibited no or a temporary response (absence of a sustained response [ASR]). For a biological risk score, five variables were selected by regularized logistic regression that predicted ASR vs CSR: (1) hemoglobin; (2) platelet count; (3) genetic polymorphisms of Fc-receptor (Fc gamma R) IIc; (4) the presence of immunoglobulin G (IgG) anti-platelet antibodies; and (5) preceding vaccination. The ASR sensitivity was 0.91 (95% confidence interval, 0.80-1.00) and specificity was 0.67 (95% confidence interval, 0.53-0.80). In the 67 patients of the observation arm, this biological score was also associated with recovery during 1 year of follow-up. The addition of the biological score to a predefined clinical score further improved the discrimination of favorable ITP disease courses.Conclusions The prediction of disease courses and IVIg treatment responses in ITP is improved by using both clinical and biological stratification.Clinical epidemiolog

A clinical prediction score for transient versus persistent childhood immune thrombocytopenia

Background Childhood immune thrombocytopenia (ITP) is an autoimmune bleeding disorder. The prognosis (transient, persistent, or chronic ITP) remains difficult to predict. The morbidity is most pronounced in children with persistent and chronic ITP. Clinical characteristics are associated with ITP outcomes, but there are no validated multivariate prediction models.Objective Development and external validatation of the Childhood ITP Recovery Score to predict transient versus persistent ITP in children with newly diagnosed ITP.Methods Patients with a diagnosis platelet count = 100 x 10(9)/L 3 months after diagnosis) versus persistent ITP. Age, sex, mucosal bleeding, preceding infection/vaccination, insidious onset, and diagnosis platelet count were used as predictors.Results In external validation, the score predicted transient versus persistent ITP at 3 months follow-up with an area under the receiver operating characteristic curve of 0.71. In patients predicted to have a high chance of recovery, we observed 85%, 90%, and 95% recovered 3, 6, and 12 months after the diagnosis. For patients predicted to have a low chance of recovery, this was 32%, 46%, and 71%. The score also predicted cessation of bleeding symptoms and the response to intravenous immunoglobulins (IVIg).Conclusion The Childhood ITP Recovery Score predicts prognosis and may be useful to individualize clinical management. In future research, the additional predictive value of biomarkers can be compared to this score. A risk calculator is available ().Clinical epidemiolog

Anti-HLA antibodies with complementary and synergistic interaction geometries promote classical complement activation on platelets

High titers of HLA antibodies are associated with platelet refractoriness, causing poor platelet increments after transfusions in a subset of patients with HLA antibodies. Currently, we do not know the biological mechanisms that explain the variability in clinical responses in HLA alloimmunized patients receiving platelet transfusions. Previously we showed that a subset of anti-HLA IgG-antibodies induces FcγRIIa-dependent platelet activation and enhanced phagocytosis. Here, we investigated whether anti-HLA IgG can induce complement activation on platelets. We found that a subset of anti-HLA IgG induced complement activation via the classical pathway, causing C4b and C3b deposition and formation of the membrane-attack complex. This resulted in permeabilization of platelet membranes and increased calcium influx. Complement activation also caused enhanced α-granule release, as measured by CD62P surface exposure. Blocking studies revealed that platelet activation was caused by FcγRIIa-dependent signaling as well as HLA antibody induced complement activation. Synergistic complement activation employing combinations of monoclonal IgGs suggested that assembly of oligomeric IgG complexes strongly promoted complement activation through binding of IgGs to different antigenic determinants on HLA. In agreement with this, we observed that preventing anti-HLA-IgG hexamer formation using an IgG-Fc:Fc blocking peptide, completely inhibited C3b and C4b deposition. Our results show that HLA antibodies can induce complement activation on platelets including membrane attack complex formation, pore formation and calcium influx. We propose that these events can contribute to fast platelet clearance in vivo in patients refractory to platelet transfusions with HLA alloantibodies, who may benefit from functional-platelet matching and treatment with complement inhibitors

Fc galactosylation of anti-platelet human IgG1 alloantibodies enhances complement activation on platelets

Approximately 20% of patients receiving multiple platelet transfusions develop platelet alloantibodies, which can be directed against human leukocyte antigens (HLA) and, to a lesser extent, against human platelet antigens (HPA). These antibodies can lead to the rapid clearance of donor platelets, presumably through IgG-Fc receptor (FcγR)-mediated phagocytosis or via complement activation, resulting in platelet refractoriness. Strikingly, not all patients with anti-HLA or -HPA antibodies develop platelet refractoriness upon unmatched platelet transfusions. Previously, we found that IgG Fc glycosylation of anti-HLA antibodies was highly variable between patients with platelet refractoriness, especially with respect to galactosylation and sialylation of the Fc-bound sugar moiety. Here, we produced recombinant glycoengineered anti-HLA and anti- HPA-1a monoclonal antibodies with varying Fc galactosylation and sialylation levels and studied their ability to activate the classical complement pathway. We observed that anti-HLA monoclonal antibodies with different specificities, binding simultaneously to the same HLA-molecules, or anti-HLA in combination with anti-HPA-1a monoclonal antibodies interacted synergistically with C1q, the first component of the classical pathway. Elevated Fc galactosylation and, to a lesser extent, sialylation significantly increased the complement-activating properties of anti-HLA and anti-HPA-1a monoclonal antibodies. We propose that both the breadth of the polyclonal immune response, with recognition of different HLA epitopes and in some cases HPA antigens, and the type of Fc glycosylation can provide an optimal stoichiometry for C1q binding and subsequent complement activation. These factors can shift the effect of a platelet alloimmune response to a clinically relevant response, leading to complement-mediated clearance of donor platelets, as observed in platelet refractoriness