Anti-erythrocyte Autoantibodies Research Articles

Initial Report of Povetacicept, an Enhanced Dual BAFF/APRIL Antagonist, in Autoimmune Cytopenias: The RUBY-4 Study

Introduction: BAFF and APRIL are cytokines with key roles in B cell development, survival, differentiation into antibody-secreting cells (ASCs; plasmablasts and plasma cells), and production of antibodies, making them promising targets for the treatment of autoantibody-associated diseases such as immune thrombocytopenia (ITP), warm autoimmune hemolytic anemia (wAIHA), and cold agglutinin disease (CAD). Patients with ITP or AIHA have elevated serum BAFF and APRIL levels and belimumab, a BAFF inhibitor, has exhibited encouraging efficacy in the treatment of lupus-associated ITP as well as alone or in combination with rituximab for the treatment of primary ITP. Due to their overlapping but non-redundant roles, inhibition of both BAFF and APRIL may be required for optimal efficacy in autoimmune cytopenias. In preclinical studies, dual BAFF/APRIL inhibition achieved superior suppression of ASCs and immunoglobulin levels vs inhibitors of BAFF or APRIL alone or an anti-CD20 antibody, suggesting the potential of this novel mechanism to modify the underlying pathogenesis of autoimmune cytopenias. Povetacicept (ALPN-303) is a TACI-Fc fusion protein engineered for potent dual BAFF/APRIL inhibition. In a mouse model of AIHA, povetacicept suppressed anti-erythrocyte autoantibodies and increased hematocrit (ASH 2022 abstract #3763). In a first-in-human study in healthy volunteers, povetacicept was well tolerated and reduced levels of circulating ASCs and immunoglobulins. This is an initial report of an open-label study of povetacicept in autoimmune cytopenias (RUBY-4; NCT05757570). Methods: RUBY-4 is a phase 1b, group-sequential, parallel-cohort study evaluating povetacicept in participants aged ≥18 years with a documented diagnosis of primary ITP, wAIHA, or CAD of ≥12 weeks in duration (Figure). Eligible participants must have sustained thrombocytopenia (ITP cohort) or sustained symptomatic anemia (wAIHA/CAD cohorts)-i.e., platelet count <30 × 10 9/L or hemoglobin ≤10 g/dL on 2 occasions ≥7 days apart during screening-and a history of failure or relapse on ≥2 prior treatments (including a thrombopoietin receptor agonist for the ITP cohort). Background therapy, including corticosteroids, is allowed but must remain at stable doses for the durations specified in the protocol. Participants receive povetacicept 240 mg subcutaneously once every 4 weeks for 24 weeks, with an optional 24-week treatment extension, followed by an 8-week off-treatment follow-up period. The primary objective is safety and tolerability; secondary objectives include pharmacokinetics, pharmacodynamics, immunogenicity, and efficacy. Results: As of 31 July 2023, 1 participant with ITP has enrolled and received 2 doses of povetacicept 240 mg without incident. Initial, preliminary observations include expected reductions in B cells and immunoglobulin levels. The RUBY-4 study continues to enroll in the US, Canada, Australia, and Turkey. At the time of presentation, available data including safety, pharmacokinetics, pharmacodynamics, and preliminary efficacy (e.g., platelet or hemoglobin response rate, duration of response, and time to response) findings will be reported. Conclusions: To our knowledge, povetacicept is the first dual BAFF/APRIL inhibitor to be evaluated for the treatment of autoimmune cytopenias. Previous preclinical and healthy volunteer studies suggest the potential of povetacicept for suppression of the pathogenic autoimmunity that mediates platelet/erythrocyte destruction in ITP, wAIHA, and CAD, which may lead to more durable responses and improved outcomes for patients.

Use of steroids in the management of low-risk myelodysplastic syndromes with autoimmune features.

The boundaries between myelodysplastic syndromes (MDS) and immune-mediated cytopenias are often difficult to establish and both conditions may benefit from immunosuppressive therapy. The optimal timing and doses of immunosuppressants are largely unknown. We systematically evaluated a retrospective cohort of 79 patients with low-risk MDS tested for anti-erythrocyte or anti-platelet autoantibodies to assess their frequency and the efficacy of immunosuppression, particularly with steroids. We found autoantibody positivity in 43% of cases and overt autoimmune diseases in 18%, including autoimmune hemolytic anemia, immune thromboctyopenia, and Evans syndrome. Steroid treatment improved cytopenia in about half of patients, with 26% achieving a complete recovery lasting for a median of 12 months. Better responses were observed in anemic patients with anti-erythrocyte autoantibodies than in those with anti-platelet autoantibodies, and the combination with recombinant erythropoietin (7/10) had a possible synergistic effect. Steroid doses were heterogeneous depending on the clinical intent (i.e., anti-inflammatory, immunosuppressive, anabolizing). Patients treated with a dose of 1 mg/kg day of prednisone for overt autoimmune cytopenia showed high rates of complete responses (60%). This observation suggests a trial with a short course (2-3 weeks) of standard steroid doses to ascertain efficacy and properly silence the autoimmune pathogenic mechanism. Steroid-related adverse events (16% of cases) should be monitored carefully in this elderly, frail population. In conclusion, features of autoimmunity are present in more than two-thirds of low-risk MDS patients and a trial with prednisone 0.5-1 mg/kg day for 2-3 weeks, with proper monitoring of adverse events, may be useful to improve cytopenias in selected cases.

The Indirect Antiglobulin (Coombs') Test Is Specific but Less Sensitive Than the Direct Antiglobulin Test for Detecting Anti-Erythrocytic Autoantibodies and Thereby Immune-Mediated Hemolytic Anemia in Dogs.

The immunodiagnostic assessment of dogs suspected of having immune-mediated hemolytic anemia (IMHA) is based on persistent autoagglutination of erythrocytes (after three saline washes), marked spherocytosis, and a positive direct antiglobulin (Coombs') test (DAT). However, the value of using the indirect antiglobulin test (IAT) for the detection of anti-erythrocytic autoantibodies in serum from dogs suspected of having IMHA is unclear. To evaluate the IAT, leftover serum samples from a large cohort of 94 dogs suspected of having IMHA and for which DAT results were known were incubated with DAT- erythrocytes, and five IAT techniques were performed (in part with different reagents and temperatures): microtiter plate (MICRO), microcapillary, laboratory gel column, gel minitube kit (GEL KIT), and immunochromatographic strip kit. Two IAT techniques (MICRO at 37 °C and GEL KIT with rabbit anti-dog polyvalent reagent) detected autoantibodies against erythrocytes in serum from 53% and 57% of DAT+ dogs, respectively, while other IATs performed less well. Moreover, while the analytic specificity of the IAT methods compared to the DAT ranged from 96-100%, the sensitivity range was only 9-57%. Thus, we still recommend DAT for diagnosis and monitoring of IMHA in dogs but conclude that a positive IAT result may aid diagnostically when serum is available, but fresh red blood cells are not.

Autoimmune hemolytic anemia: the current state of the issue

Autoimmune hemolytic anemia is a rare disease characterized by the appearance of anti-erythrocyte autoantibodies and subsequent destruction of red blood cells by cells of the immune system. The destruction mechanisms of erythrocytes loaded with autoantibodies are well studied; however, the initial mechanisms that trigger the production of antibodies to own erythrocytes antigens remain unclear. In the pathogenesis of autoimmune hemolytic anemia, an important role is played by impaired immunological tolerance, in which T-lymphocytes play a key role. The study of T-lymphocytes subpopulation in patients with autoimmune hemolytic anemia by flow cytometry can provide valuable information for studying the disease pathogenesis and developing new approaches to its treatment.

Characterization of anti-erythrocyte and anti-platelet antibodies in hemolytic anemia and thrombocytopenia induced by Plasmodium spp. and Babesiaspp. infection in mice.

Malaria and Babesiosis are acute zoonotic disease that caused by infection with the parasite in the phylum Apicomplexa. Severe anemia and thrombocytopenia are the most common hematological complication of malaria and babesiosis. However, the mechanisms involved have not been elucidated, and only a few researches focus on the possible role of anti-erythrocyte and anti-platelet antibodies. In this study, the Plasmodium yoelii, P. chabaudi, Babesia microti and B. rodhaini infected SCID and ICR mice. The parasitemia, survival rate, platelet count, anti-platelet antibodies, and the level of IFN-γ and interleukin (IL) -10 was tested after infection. Furthermore, the P. yoelii, P. chabaudi, B. rodhaini and B. microti infected ICR mice were treated with artesunate and diminaze, the development of the anti-erythrocyte and anti-platelet antibodies in chronic stage were examined. At last, the murine red blood cell and platelet membrane proteins probed with auto-antibodies induced by P. yoelii, P. chabaudi, B. rodhaini, and B. microti infection were characterized by proteomic analysis. The high anti-platelet and anti-erythrocyte antibodies were detected in ICR mice after P. yoelii, P. chabaudi, B. rodhaini, and B. microti infection. Actin of murine erythrocyte and platelet is a common auto-antigen in Plasmodium and Babesia spp. infected mice. Our findings indicate that anti-erythrocyte and anti-platelet autoantibodies contribute to thrombocytopenia and anemia associated with Plasmodium spp. and Babesia spp. infection. This study will help to understand the mechanisms of malaria and babesiosis-related thrombocytopenia and hemolytic anemia.

Autoimmune Hemolytic Anemia (AHA) with hyperglycemia in a 22-year-old woman: a case report

Background: Autoimmune hemolytic anemia (AIHA) is defined as increased erythrocyte destruction due to anti-erythrocyte autoantibodies with or without complement activation. Diabetes Mellitus (DM) is a chronic metabolic disease characterized by elevated blood glucose levels, which cause serious damage to various organs, including the heart, blood vessels, eyes, kidneys, and nerves. The impact of DM on a patient's survival with AIHA is unknown. Case Presentation: A 22-year-old woman complained of dizziness and fainting before admission. The patient looked pale; on physical examination, we found jaundice on the sclera. This patient was diagnosed with AIHA and DM with increased HbA1c (10.8%) since 2017 but did not take the anti-diabetic drugs regularly. Hematological examination showed anemia, increased reticulocytes, and a positive direct Coombs test. The result of increased fasting glucose, 2 hours OGTT, and HbA1c levels (4.6%) tests were not relevant to hyperglycemia. In this patient, we also found normal C-peptide levels. The decrease in HbA1c levels was thought to be due to hemolytic anemia; therefore, HbA1c was falsely low due to rapid erythrocyte turnover. Conclusion: This patient was diagnosed with DM based on American Diabetes Association criteria with normal C-peptide levels, suggesting Maturity Onset Diabetes of the Young with HbA1c incompatibility with glucose levels may be due to rapid erythrocyte turnover.

Low-Risk Myelodysplastic Syndrome Revisited: Morphological, Autoimmune, and Molecular Features as Predictors of Outcome in a Single Center Experience.

Low-risk myelodysplastic syndromes (LR-MDS) are a very heterogeneous disease, with extremely variable clinical features and outcome. Therapeutic strategies are still limited and mainly consist of erythropoiesis-stimulating agents (ESAs) and transfusion support. The contribution of molecular lesions and of autoimmune phenomena to pathogenesis and clinical course, including leukemic evolution, is a field of open investigation. We analyzed data from a cohort of 226 patients with LR-MDS followed at our center in the last 20 years, focusing on morphological, immunological (antiplatelets and anti-erythrocyte autoantibodies, anti-erythroblast antibodies), and molecular features. Hypoplastic bone marrow was found in 7% of the cases correlating with younger age, deeper cytopenia, lower dysplasia, and worse response to ESAs. A marker of autoimmunity was observed in 46% of the tested cases, who were younger, were less frequent dysplastic changes, and responded better to ESAs and steroids. Finally, 68% of the tested cases displayed at least one somatic mutation, most commonly SF3B1, TET2, ASXL1, and SRSF2, associated with older age, presence of neutropenia, and lower response to ESAs. Leukemic evolution (2.2%) was associated with presence of somatic mutations, and survival was favorably related to response to ESAs and transfusion independence. Overall, granular evaluation and re-evaluation are pivotal in LR-MDS patients to optimize clinical management.

A New Murine Model of Primary Autoimmune Hemolytic Anemia (AIHA).

Loss of humoral tolerance to red blood cells (RBCs) can lead to autoimmune hemolytic anemia (AIHA), a severe, and sometimes fatal disease. Patients with AIHA present with pallor, fatigue, decreased hematocrit, and splenomegaly. While secondary AIHA is associated with lymphoproliferative disorders, infections, and more recently, as an adverse event secondary to cancer immunotherapy, the etiology of primary AIHA is unknown. Several therapeutic strategies are available; however, there are currently no licensed treatments for AIHA and few therapeutics offer treatment-free durable remission. Moreover, supportive care with RBC transfusions can be challenging as most autoantibodies are directed against ubiquitous RBC antigens; thus, virtually all RBC donor units are incompatible. Given the severity of AIHA and the lack of treatment options, understanding the cellular and molecular mechanisms that facilitate the breakdown in tolerance would provide insight into new therapeutics. Herein, we report a new murine model of primary AIHA that reflects the biology observed in patients with primary AIHA. Production of anti-erythrocyte autoantibodies correlated with sex and age, and led to RBC antigen modulation, complement fixation, and anemia, as determined by decreased hematocrit and hemoglobin values and increased reticulocytes in peripheral blood. Moreover, autoantibody-producing animals developed splenomegaly, with altered splenic architecture characterized by expanded white pulp areas and nearly diminished red pulp areas. Additional analysis suggested that compensatory extramedullary erythropoiesis occurred as there were increased frequencies of RBC progenitors detectable in the spleen. No significant correlations between AIHA onset and inflammatory status or microbiome were observed. To our knowledge, this is the first report of a murine model that replicates observations made in humans with idiopathic AIHA. Thus, this is a tractable murine model of AIHA that can serve as a platform to identify key cellular and molecular pathways that are compromised, thereby leading to autoantibody formation, as well as testing new therapeutics and management strategies.

A Case of Autoimmune Hemolytic Anemia Following COVID-19 Messenger Ribonucleic Acid Vaccination

Autoimmune hemolytic anemia (AIHA) is a condition characterized by the increased destruction of red blood cells (RBCs) mediated by anti-erythrocyte autoantibodies with or without complement activation. Its clinical presentation is heterogeneous, ranging from asymptomatic to severe forms with fatal outcomes, and it can be either idiopathic or secondary to a coexisting disorder. In this report, we present a case of a patient who suffered from acute and severe AIHA after receiving the second dose of the coronavirus disease 2019 (COVID-19) messenger ribonucleic acid (mRNA) vaccine.

Can Treatment with Venetoclax for Chronic Lymphocytic Leukemia (CLL) Result In Autoimmune Hemolytic Anemia?

Patient: Female, 62-year-oldFinal Diagnosis: Autoimmune hemolytic anemiaSymptoms: AnemiaMedication:—Clinical Procedure: —Specialty: HematologyObjective:Unusual clinical courseBackground:Chronic lymphocytic leukemia (CLL) is a hematological disease characterized by the clonal proliferation and accumulation of neoplastic B lymphocytes in the blood, bone marrow, lymph nodes, and spleen. Autoimmune hemolytic anemia (AIHA) is an acquired hemolytic anemia in which the destruction of erythrocytes is helped by anti-erythrocyte auto-antibodies. This has a controversial effect on the clinical outcome and survival of patients with CLL. Venetoclax, a second-generation BH3 mimetic compound, is one of the new therapies that has been approved for the treatment of CLL. Venetoclax disrupts the antiapoptotic signaling through BCL2. Common adverse events associated with venetoclax include neutropenia, thrombocytopenia, and diarrhea. This case report describes a patient with CLL who developed AIHA when treated with venetoclax.Case Report:A patient of 62-year-old woman, who was treated with multiple lines of therapy, presented autoimmune hemolytic anemia after treatment with venetoclax. The anemia was resolved after holding venetoclax and being treated with rituximab. In January 2019, there were reports of 7 patients developing AIHA related to veneto-clax therapy in Europe, according to the EudraVigilance database. How venetoclax can cause AIHA is not completely clear. This complication can happen when the erythrocyte antigen is altered by the drug that can produce antibodies. The other described mechanism is the binding of the drug with erythrocytes, which leads to production of an immune response.Conclusions:Although AIHA can be a complication of CLL, it may be caused by treatment with venetoclax. That may be confirmed after eliminating other causes.

Reduced 25-OH vitamin D in patients with autoimmune cytopenias, clinical correlations and literature review.

Vitamin D deficiency is widespread in Western Countries and has been found related to autoimmune and hematologic disease incidence and clinical course. We evaluated vitamin D levels, vitamin D receptor (VDR) and T helper (Th)1, Th2 and Th17 immunomodulatory cytokines in patients with immune thrombocytopenic purpura (ITP, N=44), primary autoimmune hemolytic anemia (AIHA, n=35), Evans' syndrome (n=5) and chronic idiopathic neutropenia (CIN, n=19) and also tested vitamin D effect on the in vitro production of anti-erythrocyte autoantibodies. 25-OH-vitamin D levels were significantly lower and vitamin D receptor higher in patients than in controls. Among ITP cases, those with very low vitamin D levels displayed reduced platelet counts, irrespective of the bleeding history. In AIHA patients, LDH values negatively correlated with vitamin D levels in mixed forms, and reticulocyte counts were positively related with vitamin D. Considering treatment, AIHA patients who had been treated with 2 therapy lines or more showed lower mean 25-OH-vitamin D levels than those untreated or treated with one line of therapy only. IL-6, IL-10, IL-17 and IFN-γ levels were higher in patients versus controls, whereas TNF-α was significantly reduced. Finally, vitamin D at concentrations of 10, 20, and 40ng/mL reduced the in vitro production of anti-erythrocyte autoantibodies both in pokeweed-stimulated and unstimulated cultures. In conclusion, vitamin D is reduced in autoimmune cytopenias and correlate with disease severity, supporting its possible protective role against the development of autoimmunity. Literature review showed vitamin D deficiency reports both in onco- and in non onco-hematologic diseases with a relationship with disease severity/activity in myeloid and lymphoid neoplasms, as well as in sickle cell disease. Supplementation has produced weak results in autoimmune and hematologic diseases, and further studies are needed.

Involvement of Fcα/μ Receptor in IgM Anti-Platelet, but Not Anti-Red Blood Cell Autoantibody Pathogenicity in Mice.

IgM anti-mouse platelet autoantibodies cause thrombocytopenia by mediating uptake of opsonized thrombocytes, whereas IgM anti-erythrocyte autoantibodies induce anemia through a phagocytosis-independent cell destruction. In this article, we show that infection with lactate dehydrogenase-elevating virus, a benign mouse arterivirus, exacerbates the pathogenicity of IgM anti-platelet, but not anti-erythrocyte autoantibodies. To define the role of Fcα/μ receptor (Fcα/μR) in IgM-mediated thrombocytopenia and anemia, we generated mice deficient for this receptor. These animals were resistant to IgM autoantibody-mediated thrombocytopenia, but not anemia. However, the lactate dehydrogenase-elevating virus-induced exacerbation of thrombocytopenia was not associated with enhanced Fcα/μR expression on macrophages. These results indicate that Fcα/μR is required for the pathogenicity of IgM anti-platelet autoantibodies but is not sufficient to explain the full extent of the disease in virally infected animals.

Complement deposition in autoimmune hemolytic anemia is a footprint for difficult-to-detect IgM autoantibodies.

In autoimmune hemolytic anemia autoantibodies against erythrocytes lead to increased clearance of the erythrocytes, which in turn results in a potentially fatal hemolytic anemia. Depending on whether IgG or IgM antibodies are involved, response to therapy is different. Proper identification of the isotype of the anti-erythrocyte autoantibodies is, therefore, crucial. However, detection of IgM autoantibodies can be challenging. We, therefore, set out to improve the detection of anti-erythrocyte IgM. Direct detection using a flow cytometry-based approach did not yield satisfactory improvements. Next, we analyzed whether the presence of complement C3 on a patient's erythrocytes could be used for indirect detection of anti-erythrocyte IgM. To this end, we fractionated patients' sera by size exclusion chromatography and tested which fractions yielded complement deposition on erythrocytes. Strikingly, we found that all patients with C3 on their erythrocytes according to standard diagnostic tests had an IgM anti-erythrocyte component that could activate complement, even if no such autoantibody had been detected with any other test. This also included all tested patients with only IgG and C3 on their erythrocytes, who would previously have been classified as having an IgG-only mediated autoimmune hemolytic anemia. Depleting patients' sera of either IgG or IgM and testing the remaining complement activation confirmed this result. In conclusion, complement activation in autoimmune hemolytic anemia is mostly IgM-mediated and the presence of covalent C3 on patients' erythrocytes can be taken as a footprint of the presence of anti-erythrocyte IgM. Based on this finding, we propose a diagnostic workflow that will aid in choosing the optimal treatment strategy.

Lack of Galactosylation Enhances the Pathogenic Activity of IgG1 but Not IgG2a Anti-Erythrocyte Autoantibodies

IgG bears asparagine-linked oligosaccharide side chains in the Fc region. Variations in their extent of galactosylation and sialylation could modulate IgG Fc-dependent effector functions, and hence Ab activity. However, it has not yet been clarified whether the pathogenic potential of IgG autoantibodies is consistently enhanced by the absence of galactose residues per se or the lack of terminal sialylation, which is dependent on galactosylation. Moreover, it remains to be defined whether the increased pathogenicity of agalactosylated IgG is related to activation of the complement pathway by mannose-binding lectin, as suggested by in vitro studies. Using a murine model of autoimmune hemolytic anemia, we defined the contribution of galactosylation or sialylation to the pathogenic activity of IgG1 and IgG2a anti-erythrocyte class-switch variants of 34-3C monoclonal autoantibody. We generated their degalactosylated or highly sialylated glycovariants and compared their pathogenic effects with those of highly galactosylated or desialylated counterparts. Our results demonstrated that lack of galactosylation, but not sialylation, enhanced the pathogenic activity of 34-3C IgG1, but not IgG2a autoantibodies. Moreover, analysis of in vivo complement activation and of the pathogenic activity in mice deficient in C3 or IgG FcRs excluded the implication of mannose-binding lectin-mediated complement activation in the enhanced pathogenic effect of agalactosylated IgG1 anti-erythrocyte autoantibodies.

Generation of monoclonal autoantibodies from Babesia rodhaini-infected mice.

ABSTRACTThe presence of anti-erythrocyte autoantibodies in animals infected with various Babesia species is well reported. However, the pathogenesis of autoantibodies in babesiosis is poorly understood. Here, we demonstrated that anti-erythrocyte immunoglobulin (Ig) M and IgG were present in B. rodhaini-infected mice at 6 and 8 days after infection, respectively. Furthermore, we generated monoclonal antibodies against erythrocyte antigen from B. rodhaini-infected mice. Five clones were generated. By Western blotting analysis using whole erythrocyte antigens, one clone reacted with a broad band around 90–150 kDa, and the 2 clones reacted with a band larger than 150 kDa. B. rodhaini-infected mice and/or autoreactive monoclonal antibodies established in this study might be a powerful tool for in vivo pathogenesis studies of autoantibody development in infectious diseases.

Clinical Impact Of The Simultaneous Detection Of Anti-Erythocytic Autoantibodies In Alloimmunization

Introduction The simultaneous detection of autoantibodies and alloantibodies to red blood cells (RBC) following allogeneic transfusion is poorly understood. The incidence of autoimmune hemolysis in these patients is not well established (Young et al., Transfusion 2004; 44: 67-72). Methods Patients with a positive antibody screen, an identified alloantibody and a positive direct antiglobulin test (DAT) were selected from our hospital Blood Bank database from January 2002 to June 2013. Sex, age, main clinical diagnosis, transfusion history, alloantibody and autoantibody specificity and hemolysis parameters were analysed retrospectively in those patients with simultaneous first detection of auto and alloantibodies. In our Blood Bank, all patients to be transfused underwent a three-cell antibody screen by gel technology (Ortho Clinical Diagnosis). If the screen was positive, two microcolumn panels with eleven cells (DianaGel) and an autocontrol were performed in Liss-Coombs and with papain-treated erythrocytes for antibody identification. Test-tube testing at different temperatures with Reagent RBC (Makropanel) and a polyethyleneglycol (PEG) antiglobulin technique were carried out to confirm antibody specificity when deemed appropriate. Polyspecific and monospecific (anti-IgG and C3d) DAT (Menarini reagents) and a 6% albumin negative control were performed in all patients presenting a positive autocontrol. Acid glycine elution (Gamma ELU-KIT II) was the method of choice to prepare the eluates. Adsorption techniques were used in some cases to remove autoantibodies. Results A total of 2915 patients formed alloantibodies; 4.5% (n=132) presented both auto- and alloantibodies. The temporal relationship between the antibodies detected had the following distribution: 96.2% (n=127) simultaneous auto- and alloantibody, 2.3% alloantibody identification and on subsequent studies autoantibody and 1.5% had autoantibody previous to alloimmunization. The following results will refer to the group with simultaneous detection of auto and alloantibodies. In the selected group, 55% had a complete register of previous RBC transfusion with a median of 8.5 transfusions lifelong (range: 2 to 81) and a median of 38.5 days (range: 4 to 270 days) between last negative and first positive antibody screen. The ratio male to female was 1:1 and the median age was 67 years-old (range: 20 to 91 years-old). The most common diagnosis in these patients were: solid neoplasia (21.3%), oncohematologic disease (13.4%; mielodysplastic syndromes were included in this subgroup), chronic hepatopathy (15%; of which 58% are HVC-related), cardiovascular disease (9.5%), traumatism (7%), pregnancy (3.2%) and autoimmune disease (3.1%). The most frequent alloantibodies identified were: anti-E (39.3%) > anti-C (28.3%) > anti-K (24.5%). Most autoantibodies had no apparent specificity (67%), those that did have a specific pattern were related to Rh antigens (anti-e 7%; anti-c 1.5%; anti-D 1.5%), and the remainder 23% were crioagglutinins. A total of 16 patients (12.5%) had concomitant hemolysis at the time of antibody detection of which 43.7% (n=7) had an active cancer (57% due to oncohematologic disease), 25% (n=4) HCV hepatopathy and 12.5% autoimmune hepatitis (n=2). Conclusion Patients with de novo alloimmunization showed concomitant antierythrocytic autoantibodies in 4.5% of our population. Most cases appear to be related to previous RBC transfusion and medical conditions affecting normal immunological functioning. However, hemolysis was only found in 12.5% of these patients at the time of antibody detection and all of them had diseases with a known associated risk of developing autoimmune anemia. Disclosures: No relevant conflicts of interest to declare.

Essential requirement for IRF8 and SLC15A4 implicates plasmacytoid dendritic cells in the pathogenesis of lupus

In vitro evidence suggests that plasmacytoid dendritic cells (pDCs) are intimately involved in the pathogenesis of lupus. However, it remains to be determined whether these cells are required in vivo for disease development, and whether their contribution is restricted to hyperproduction of type I IFNs. To address these issues, we created lupus-predisposed mice lacking the IFN regulatory factor 8 (IRF8) or carrying a mutation that impairs the peptide/histidine transporter solute carrier family 15, member 4 (SLC15A4). IRF8-deficient NZB mice, lacking pDCs, showed almost complete absence of anti-nuclear, anti-chromatin, and anti-erythrocyte autoantibodies, along with reduced kidney disease. These effects were observed despite normal B-cell responses to Toll-like receptor (TLR) 7 and TLR9 stimuli and intact humoral responses to conventional T-dependent and -independent antigens. Moreover, Slc15a4 mutant C57BL/6-Fas(lpr) mice, in which pDCs are present but unable to produce type I IFNs in response to endosomal TLR ligands, also showed an absence of autoantibodies, reduced lymphadenopathy and splenomegaly, and extended survival. Taken together, our results demonstrate that pDCs and the production of type I IFNs by these cells are critical contributors to the pathogenesis of lupus-like autoimmunity in these models. Thus, IRF8 and SLC15A4 may provide important targets for therapeutic intervention in human lupus.

Clinical Relevance of an Extended Diagnostic Work-Out in Patients with Primary Autoimmune Amolytic Anemia (AIHA)

AbstractAbstract 3192Autoimmune hemolytic anemia (AIHA) is a rare disease, with an incidence of 1:100,000 for warm AIHA and of 1:1,000,000 for cold AIHA. AIHA can occur without any evidence of an underlying disorder (idiopathic or primary AIHA) or can be diagnosed in association with another disease (secondary AIHA), such as a malignancy, a lymphoproliferative disorder (LD), an autoimmune disorder, an infection. A successful treatment of secondary AIHA is based upon the treatment of the associated disease. In particular, the detection of a possible underlying LD is a relevant issue since a successful treatment approach for both conditions includes monoclonal antibodies that target B lymphocytes producing the anti-erythrocyte autoantibody (AeAb). With the aim of unravelling the presence of an associated disease, between January 2000 and January 2012, patients with an AIHA diagnosis defined as “primary” on clinical grounds and observed at our institution were included in this study. Patients were required to show anemia, serological evidence of an AeAb by a positive direct antiglobulin test, clinical and laboratory signs of hemolysis, no previous history and/or clinical signs of an associated malignancy, LD, autoimmune disorder, infection or a prior use of drugs commonly implicated in the pathogenesis of AIHA. Before starting treatment, a diagnostic work-out including the following examinations was carried out: autoantibody profile (anti-nucleus; anti-cardiolipin; anti-gastric parietal cells; anti-beta 2-glycoprotein-I; anti-peroxidase and anti-thyroglobulin); HBV, HCV and HIV serology; total body CT scan or chest X-ray associated with an abdomen ultrasound, bone marrow (BM) aspirate and biopsy, peripheral blood (PB) and BM immunophenotype by 4 color flow-cytometry analysis (CD19/CD5/CD3/CD20; CD23/CD10/CD19/CD20; Igκ/Igλ; CD19/CD5; CD3/CD4/CD45/CD8). Sixty-four patients were included in the study, 23 males and 41 females with a median age of 65.5 years (range: 20–83). The median Hb value was 7.9 g/dl (range: 4–11 g/dl), the median value of indirect bilirubin 2.2 mg/dl. The AeAb isotype form was an IgG in 38 cases (59%), IgM in 25 (39%; cold IgM associated with IgG, 4 cases; warm IgM, 1 case) and IgA in 1. After the initial screening, a primary AIHA diagnosis was confirmed in 38 cases (59%), while in 21 patients (33%) an underlying LD was detected, in 2 (3%) a cancer (breast, larynx), in 2 (3%) an autoimmune disorder (Hashimoto’s thyroiditis, athrophic gastritis), in 1 an active HCV hepatitis. In the majority of cases (19/21, 90.5%) the diagnosis of LD was made on the basis of the BM biopsy. In all cases with BM involvement, PB flow-cytometry revealed the presence of monoclonal B lymphocytes (median number of clonal lymphocytes, 0.405×109/L) displaying most frequently a lymphoma-like phenotype (CD5−/CD20+/CD23±). As expected, the group of patients with the evidence of a LD included a significantly higher rate of cases with an IgM AeAb as compared to the group with “true” primary AIHA (67% vs 12.5%; p= .005). Taken together, the results of this study demonstrate that in 41% of patients with an AIHA defined as primary on clinical grounds, an extended diagnostic work-out allowed to identify an associated disease, most frequently a LD involving the BM associated with a clonal B population in PB. These data suggest that in patients with a diagnosis of AIHA, an extended screening including a BM biopsy and a PB immunothenotype should be considered in order to identify (or exclude) the presence of a LD and to address the appropriate treatment. Disclosures:No relevant conflicts of interest to declare.

Innate sensors for nucleic acids and lupus pathogenesis

We continue our efforts to define the pathogenesis of systemic lupus-like autoimmunity in predisposed mouse models by focusing on the role of endosomal nucleic acid-sensing TLRs. We reported that NZB mice deficient for the common receptor for type I IFNs showed significant reductions in all disease parameters. To differentiate whether the pathogenic effects were mediated by the multiple IFNα subtypes and/or the single IFNβ, we created congenic NZB mice lacking Ifnb and found that disease severity was unaltered, strongly implicating IFNα subtypes as the principal effectors. We then documented that long-term treatment of male BXSB mice with an anti-IFNAR antibody of mouse origin reduced serologic, cellular and histologic disease manifestations and extended survival, suggesting that disease acceleration by the Tlr7 gene duplication in this model is mediated by type I IFN signaling. The efficacy of this treatment was greater when applied relatively early in the disease process, but reductions in some disease characteristics, especially kidney pathology, were evident even when treatment was initiated at later stages, and a transient therapeutic effect was also noted in the MRL-Faslpr model. The combined findings suggest that antibody-mediated IFNAR blockade may be a useful treatment approach in human SLE and probably other autoimmune syndromes in which these cytokines appear to play a pathogenic role. We have also hypothesized that engagement of nucleic acid-sensing TLRs may be responsible for spreading the aberrant response beyond ANAs to encompass the broader spectrum of disease-associated autoantibody specificities. Using MRL-Faslpr mice congenic for the 3d mutation of the Unc93b1 gene, in which signaling by all endosomal TLRs (TLR3, TLR7, TLR9) is extinguished, we indeed found reductions not only in autoantibodies against nucleic acids and associated proteins (anti-chromatin, anti-RNP, anti-Sm), but also in a broad panel of autoantibody specificities, particularly against antigens known to contain or bind to nucleic acids, such as cardiolipin, β2-glycoprotein 1 and myeloperoxidase. Surprisingly, even anti-erythrocyte autoantibodies and hemolytic anemia were significantly reduced in NZB mice congenic for the 3d mutation compared with wild-type controls. Thus, almost the entire gamut of autoantibodies in lupus can be traced to the initial engagement of nucleic acid-sensing TLRs. To examine whether engagement of B-cell intrinsic nucleic acid-sensing TLRs is required for autoantibody production in vivo, we generated mixed bone marrow chimeras of B6-Faslpr mice that were either 3d/IgHb or WT/IgHa and measured allotype-specific IgM RF and IgG2a anti-chromatin responses. Strikingly, both autoantibodies were derived almost exclusively from the wild-type donor B cells. This finding underscores the essential significance of B-cell nucleic acid sensors in loss of tolerance and production of autoantibodies in lupus and provides a specific target for intervention. Evidence suggests that plasmacyotoid dendritic cells (pDC), the major producers of type I IFNs, are likely to be involved in the pathogenesis of lupus. One ENU phenovariant identified by Beutler and colleagues, named feeble, showed abrogation of both TLR7-induces and TLR9-induced type I IFN and proinflammatory cytokine production by pDCs, while leaving intact pDC development and TLR responses by other cells. The feeble phenotype was mapped to a mutation in Slc15a4, which encodes the peptide/histidine transporter 1 (PHT1), one of the four members of the solute carrier 15 (Slc15) family of proteins. In preliminary collaborative studies with Beutler and colleagues, lupus-prone B6Faslpr mice congenic for the feeble mutation showed significant reduction in hypergammaglobulinemia, lymphadenopathy and mortality. These findings provide direct evidence for the role of pDC in the pathogenesis of systemic autoimmunity and suggests that pharmacologic agents that interfere with Sle15a4 function may be useful in treating lupus and other diseases in which these cells appear to be involved.

Regulatory T cells essential to prevent the loss of self-tolerance in murine models of erythrocyte-specific autoantibody responses

The spontaneous appearance of anti-erythrocyte autoantibodies resulting in autoimmune hemolytic anemia described in NZB mice more than 40years ago provided a model for the study of mechanisms behind the loss of self-tolerance. We developed an in vitro model of this anti-MRBC response in which CD8(+) suppressor T cells were shown to be a controlling element. CD8(+) T cells from young NZB mice co-cultured with spleen cells from old, actively autoimmune NZB mice suppressed the anti-MRBC responses of the old mice. Eliminating the CD8(+) cells from young NZB spleen cells or even from non-autoimmune BALB/c spleen cells prior to culture removed the controlling influence of these CD8(+) cells and allowed the development of anti-MRBC-secreting cells. This review will consider the role of the CD8(+) suppressive cells in the anti-self-erythrocyte model in light of insights provided by current 'regulatory T cell' literature.