Self-reactive IgG Research Articles

Antiviral and autoimmune antibody levels for two years post SAR-CoV-2 infection show uniquely durable vaccination responses and partial waning of self-reactive IgG

Abstract As temporal distance from the start of the COVID-19 pandemic increases, there is mounting concern of the long-term immunological consequences of SARS-CoV-2 infection(s). Abnormally high levels of a panoply of autoantibodies are found during acute COVID-19, and to date it is still unclear if these antibodies are sustained for a year or longer post-infection. Additionally, if there is improved longevity of vaccine-induced antibodies when administered against the immune backdrop of previous SARS-CoV-2 infection remains to be elucidated. To address these questions, we measured a series of anti-viral and autoimmune antibodies from samples taken at intervals from seven subjects infected with SARS-CoV-2 in the spring of 2020 (five outpatient and two hospitalized). Blood draws were performed beginning at acute disease and for an additional 10–25 months. Plasma IgG and IgA antibodies reactive with SARS-CoV-2 spike, RBD, and nucleocapsid, as well as CMV Mosaic, EBV GP 350, and Flu H1 were measured using the BU ELISA protocol, autoantibodies to self-antigens associated with connective tissue disease were measured using a commercial kit. Anti-viral antibody responses induced by vaccination were sustained for more than one year post SARS-CoV-2 infection in multiple subjects. Autoantibody trajectories varied, with many positive ‘hits’ falling below baseline after 1 year, and others remaining high for all time points measured. Future work will include increasing subject numbers to a larger cohort with differing clinical presentations and outcomes to define connections between serological signatures, protection from re-infection, and PASC. Supported by grants from CTSI Pilot grant (NIH/NCATS 5UL1TR001430-05), BU Internal Funding, MassCPR PASC Subaward #FP-0034311, and NIH R01 AG065050-01A1

Cosmc deficiency causes spontaneous autoimmunity by breaking B cell tolerance.

Factors regulating the induction and development of B cell–mediated autoimmunity are not well understood. Here, we report that targeted deletion in murine B cells of X-linked Cosmc, encoding the chaperone required for expression of core 1 O-glycans, causes the spontaneous development of autoimmune pathologies due to a breakdown of B cell tolerance. BC-CosmcKO mice display multiple phenotypic abnormalities, including severe weight loss, ocular manifestations, lymphadenopathy, and increased female-associated mortality. Disruption of B cell tolerance in BC-CosmcKO mice is manifested as elevated self-reactive IgM and IgG autoantibodies. Cosmc-deficient B cells exhibit enhanced basal activation and responsiveness to stimuli. There is also an elevated frequency of spontaneous germinal center B cells in BC-CosmcKO mice. Mechanistically, loss of Cosmc confers enhanced B cell receptor (BCR) signaling through diminished BCR internalization. The results demonstrate that Cosmc, through control of core 1 O-glycans, is a previously unidentified immune checkpoint gene in maintaining B cell tolerance.

Individual and stable autoantibody repertoires in healthy individuals

In the era towards precision medicine, we here present the individual specific autoantibody signatures of 193 healthy individuals. The self-reactive IgG signatures are stable over time in a way that each individual profile is recognized in longitudinal sampling. The IgG autoantibody reactivity towards an antigen array comprising 335 protein fragments, representing 204 human proteins with potential relevance to autoimmune disorders, was measured in longitudinal plasma samples from 193 healthy individuals. This analysis resulted in unique autoantibody barcodes for each individual that were maintained over one year’s time. The reactivity profiles, or signatures, are person specific in regards to the number of reactivities and antigen specificity. Two independent data sets were consistent in that each healthy individual displayed reactivity towards 0–16 antigens, with a median of six. Subsequently, four selected individuals were profiled on in-house produced high-density protein arrays containing 23,000 protein fragments representing 14,000 unique protein coding genes. Based on a unique, broad and deep longitudinal profiling of autoantibody reactivities, our results demonstrate a unique autoreactive profile in each analyzed healthy individual. The need and interest for broad-ranged and high-resolution molecular profiling of healthy individuals is rising. We have here generated and assessed an initial perspective on the global distribution of the self-reactive IgG repertoire in healthy individuals, by investigating 193 well-characterized healthy individuals.HighlightsA unique longitudinal profiling of autoantibody repertoires in healthy individualsAutoantibody profiles are highly individual and stable over timeAll individuals display IgG binding to human protein fragmentsThe specificity of disease associated autoantigens needs to be thoroughly characterizedThe identification of a small set of highly reactive autoantigensImportance of stringent antigen and sample specific cut-offs for defining reactivity

Curiouser and curiouser: The role(s) of AID expression in self‐tolerance

Aicda is crucial for antibody diversification by mediating Ig class-switch recombination, V(D)J hypermutation (SHM) and, in some species, gene conversion. Recently, evidence has accumulated to show that Aicda is expressed during B-cell development and that this expression in some unknown way, mediates tolerance in immature and transitional B cells. In this issue of the European Journal of Immunology, Umiker et al. [Eur. J. Immunol. 2014. 44: 3093-3108] show that enforced expression of Aicda during early B-cell development is associated with self-tolerance. Curiously, constitutive Aicda expression that begins early in B cells suppresses the generation of autoreactive IgM but promotes the expression of self-reactive IgG. In contrast, when Aicda is activated later in B-cell development, self-reactive IgM is abundant but IgG is not. These observations suggest pathways for self-tolerance that have been little explored.

Abstract 1864: Reptin as a tumor-associated antigen of epithelial ovarian cancer

Abstract Background: Reptin is also known as RuvBl2, Tip48, TIP49b, ECP51 and CGI-46, is a member of AAA+ (ATPases associated with diverse cellular activities) superfamily. Reptin is associated with cell viability and development of S. cerevisiae, D. melanogaster, X. laevis, or D. rerio has a lot of functions including transcriptional regulation, DNA damage repair and telomerase activity. Self-reactive IgG autoantibodies are present on human sera.Each self-reactive IgG autoantibody derived from pre- and post-treatment of ovarian cancer patients is competitively combined with cancer tissue gotten during the operation, self-reactive IgG autoantibodies bound to tumor antigens. As a result, self-reactive IgG autoantibodies may be decreased after treatment. In this study, we have identified and investigated the function of Reptin as an available therapeutic biomarker for ovarian cancer using difference of self-reactive IgG autoantibody amounts. Method: We carried out the two-dimensional differential gel electrophoresis analysis of immunoprecipitated tumor antigens (2D-DITA) to compare with expression level of autoantibodies in pre- and post-treatment sera of 14 ovarian cancer patients. Autoantibody differentiated between pre- and post-treatment sera was identified by MALDI-TOF. We performed qRT-PCR with 8 human ovarian surface epitheliums (HOSEs) and 14 ovarian cancer cell lines for validation studies. Reptin was also detected in other ovarian cancer tissues by immunohistochemistry (IHC). We constructed SKOV-3 cell line for overexpression Reptin using lentiviral system. For investigating function of Reptin, we performed proliferation, wound healing, and invasion assay. Results: We identified Reptin as a tumor-associated antigen of epithelial ovarian cancer. Relative mRNA level of Reptin showed higher levels in ovarian cancer cell lines than in HOSEs. Reptin protein levels were significantly upregulated in ovarian cancer tissues than normal, benign, and borderline ovarian tumors. In SKOV-3 cells, overexpression of Reptin induced inhibition of cell proliferation (P<0.0001), migration and invasion (P<0.0001). Conclusion: Our results demonstrate that Reptin is a tumor -associated antigen of ovarian cancer, i.e. tumor suppressor. Citation Format: Eun-ju Lee, Hanbyoul Cho, Ha-Yeon Shin, Wookyeom Yang, Hyunja Kwon, Sol Kim, Jae-Hoon Kim. Reptin as a tumor-associated antigen of epithelial ovarian cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1864. doi:10.1158/1538-7445.AM2014-1864

Natural IgG Autoantibodies Are Abundant and Ubiquitous in Human Sera, and Their Number Is Influenced By Age, Gender, and Disease

The presence of self-reactive IgG autoantibodies in human sera is largely thought to represent a breakdown in central tolerance and is typically regarded as a harbinger of autoimmune pathology. In the present study, immune-response profiling of human serum from 166 individuals via human protein microarrays demonstrates that IgG autoantibodies are abundant in all human serum, usually numbering in the thousands. These IgG autoantibodies bind to human antigens from organs and tissues all over the body and their serum diversity is strongly influenced by age, gender, and the presence of specific diseases. We also found that serum IgG autoantibody profiles are unique to an individual and remarkably stable over time. Similar profiles exist in rat and swine, suggesting conservation of this immunological feature among mammals. The number, diversity, and apparent evolutionary conservation of autoantibody profiles suggest that IgG autoantibodies have some important, as yet unrecognized, physiological function. We propose that IgG autoantibodies have evolved as an adaptive mechanism for debris-clearance, a function consistent with their apparent utility as diagnostic indicators of disease as already established for Alzheimer’s and Parkinson’s diseases.

Toll like receptor 7- and 8-dependent double-stranded DNA antibodies in a Lupus mouse model

A recognizing self RNA and dsDNA are implicated in the pathogenesis of systemic lupus erythematosus (SLE). Anti-RNA and anti-dsDNA IgG are thought to be TLR 7/8and TLR 9-dependent, respectively. Paradoxically, recent results show TLR 9 is protective in murine models of SLE, calling into question the pathogenicity and/or the mechanism of anti-dsDNA antibody generation. We study SLE “564Igi” mice that express knocked-in immunoglobulin (Ig) heavy (H) and light (L) chain genes that encode an RNAspecific antibody. Our preliminary results indicate that aged 564Igi mice shift their antibody repertoire from anti-RNA IgG to antidsDNA IgG. Surprisingly, the production of this anti-dsDNA antibody is TLR 7/8-dependent and TLR 9-independent. Our hypothesis is that these anti-dsDNA antibodies arise via somatic hypermutation (SHM) of Ig genes in B cells that are activated via the RNA-sensing TLR 7/8 pathway yet are polyreactive to dsDNA in addition to RNA. This work defines a novel mechanism for the generation of selfreactive IgG antibodies and may explain why RNA reactivity precedes polyreactivity in SLE, as is suggested by human data.

The inflammatory cytokine IL-18 induces self-reactive innate antibody responses regulated by natural killer T cells

Inflammatory responses initiate rapid production of IL-1 family cytokines, including IL-18. This cytokine is produced at high levels in inflammatory diseases, including allergy and autoimmunity, and is known to induce IgE production in mice. Here we provide evidence that IL-18 is directly coupled to induction of self-reactive IgM and IgG antibody responses and recruitment of innate B2 B cells residing in the marginal zone of the spleen. Moreover, the data suggest that the B-cell activation occurs predominantly in splenic extrafollicular plasma cell foci and is regulated by natural killer T (NKT) cells that prevent formation of mature germinal centers. We also find evidence that NKT cells control this type of B-cell activation via cytotoxicity mediated by both the perforin and CD95/CD178 pathways. Thus, NKT cells regulate innate antibody responses initiated by an inflammatory stimulus, suggesting a general mechanism that regulates B-cell behavior in inflammation and autoreactivity.

Differential regulation of self-reactivity discriminates between IgG+human circulating memory B cells and bone marrow plasma cells

Long-term humoral immunity is maintained by the formation of high-affinity class-switched memory B cells and long-lived antibody-secreting plasma cells. In healthy humans, a substantial fraction of IgG-positive memory B cells express self-reactive and polyreactive IgG antibodies that frequently develop by somatic mutations. Whether self- and polyreactive IgG-secreting B cells are also tolerated in the long-lived plasma cell pool is not known. To address this question, we cloned and expressed the Ig genes from 177 IgG-producing bone marrow plasma cells of four healthy donors. All antibodies were highly mutated but the frequency of self- and polyreactive IgG antibodies was significantly lower than that found in circulating memory B cells. The data suggest that in contrast to the development of memory B cells, entry into the bone marrow plasma cell compartment requires previously unappreciated selective regulation by mechanisms that limit the production of self- and polyreactive serum IgG antibodies.

CTLA4Ig Alters the Course of Autoimmune Disease Development in Lyn−/− Mice

Lyn-deficient (Lyn(-/-)) mice develop an age-dependent autoimmune disease similar to systemic lupus erythematosus, characterized by the production of IgG anti-nuclear Ab. To determine the extent to which this autoimmune phenotype is driven by T cell costimulation, we generated Lyn(-/-) mice expressing a soluble form of the T cell inhibitory molecule, CTLA4 (CTLA4Ig). Surprisingly, although CTLA4Ig prevented myeloid hyperplasia, splenomegaly and IgG anti-nuclear Ab production in Lyn(-/-) mice, it did not inhibit immune complex deposition and tissue destruction in the kidney. In fact, regardless of CTLA4Ig expression, Lyn(-/-) serum contained elevated titers of IgA anti-nuclear Ab, although generally IgA deposition in the kidney was only revealed in the absence of self-reactive IgG. This demonstrated that activation of autoreactive B cell clones in Lyn(-/-) mice can still occur despite impaired costimulation. Indeed, CTLA4Ig did not alter perturbed Lyn(-/-) B cell development and behavior, and plasma cell frequencies were predominantly unaffected. These results suggest that when self-reactive B cell clones are unimpeded in acquiring T cell help, they secrete pathogenic IgG autoantibodies that trigger the fulminant autoimmunity normally observed in Lyn(-/-) mice. The absence of these IgG immune complexes reveals an IgA-mediated axis of autoimmunity that is not sufficient to cause splenomegaly or extramedullary myelopoiesis, but which mediates destructive glomerulonephritis. These findings have implications for the understanding of the basis of Ab-mediated autoimmune diseases and for their treatment with CTLA4Ig.

Identification of target antigens of self‐reactive IgG in intravenous immunoglobulin preparations

Intravenous immunoglobulin (IVIg) contains a wide range of self-reactive immunoglobulins (Ig) G. Acidic pH is known to increase the reactivity of purified IgG with self-antigens. We describe here the target antigens of IgG autoantibodies in IVIg and analyze the influence of acidic pH on IgG reactivities. We used 2-DE and immunoblotting with protein extracts of human umbilical vein endothelial cells (HUVEC) and HEp-2 cells. Two IVIg preparations obtained by ethanol fractionation [one with an acidic pH step (acidic-IVIg) and one with beta-propiolactone (propiolactone-IVIg)] and a pool of sera from 12 healthy individuals were tested. Serum IgG of 3 healthy individuals and IgG purified from the same sera with elution at pH 2.8 were also tested individually. Finally, propiolactone-IVIg was acidified at pH 2.8. IgG obtained with a step at low pH recognized many more target spots than IgG obtained without acidic pH. Our findings demonstrate that an acidic pH step artificially enlarges the repertoire of self-reactive IgG. Thus, protein spots recognized by IgG in propiolactone-IVIg represent the core set of self-antigens targeted by IVIg. Overall, 96 proteins were identified by MS. Fourteen were recognized in both extracts including glycolysis proteins such as alpha-enolase, RNA processing and cytoskeletal proteins such as lamin-A/C.

Serum IgG repertoire in clinically isolated syndrome predicts multiple sclerosis

Objective We previously showed that serum IgG repertoires distinguished multiple sclerosis (MS) patients from healthy subjects and from patients with other inflammatory neurological diseases (OIND). We questioned whether the serum IgG repertoire of patients presenting a clinically isolated syndrome (CIS) could predict MS. Methods The global IgG immune responses against brain antigens in sera from 50 CIS patients were evaluated by immunoblotting. The IgG reactivities were compared with those from MS sera (n = 82), healthy sera (n = 27), and sera from OIND (n = 42). A linear discriminant analysis (LDA) defined a score for each individual. Results About 78% of scores obtained from CIS patients were located in the “MS area.” During the follow-up (3.5 ± 1.3 years), 28 patients fulfilled the McDonald criteria for MS, 15 patients remained CIS, and 7 patients developed OIND. Among the patients with an LDA score in the “MS area,” 61.5% converted to MS. Discussion Our results suggest that a pathological distortion of the self-reactive IgG repertoire occurs early so that immunomodulating treatment should be started as early as possible; they also highlight the early involvement of B cells in the physiopathological process in MS.

Autoreactivity in Human IgG+ Memory B Cells

More than half of the nascent B cells in humans initially express autoreactive antibodies. However, most of these autoantibodies are removed from the repertoire at two checkpoints before maturation into naive B cells. A third checkpoint excludes remaining autoantibodies from the antigen-experienced IgM(+) memory B cell pool. Nevertheless, low-affinity self-reactive antibodies are frequently found in the serum of normal humans. To determine the source of these antibodies, we cloned and expressed antibodies from circulating human IgG(+) memory B cells. Surprisingly, we found that self-reactive antibodies including anti-nuclear antibodies were frequently expressed by IgG(+) memory B cells in healthy donors. Most of these antibodies were created de novo by somatic hypermutation during the transition between mature naive and IgG(+) memory B cells. We conclude that deregulation of self-reactive IgG(+) memory B cells may be associated with autoimmunity.

Changes in self-reactive IgG antibody repertoire after treatment of experimental autoimmune encephalomyelitis with anti-allergic drugs

We reduced EAE severity by using two anti-allergic drugs. A control group of mice received i.p. injections of PBS as vehicle while a further two groups were treated either with pyrilamine, a histamine receptor 1 antagonist or with CV6209, a platelet activating factor receptor antagonist. Our results showed that the blockade of the responses to both histamine and PAF leads together to a decline in clinical signs of EAE and significant changes in the serum IgG recognition of some healthy brain antigenic targets. We characterized two discriminant antigens: internexin neuronal intermediate filament protein, and malate dehydrogenase 1, which were able to clearly distinguish untreated mice from treated mice. Their role as potent targets in pathogenic and/or neuroprotective processes is discussed.

Warm autoimmune hemolytic anemia is an IgM–IgG immune complex disease

Warm autoimmune hemolytic anemia (WAIHA) is characterized by polyclonal IgG autoantibodies binding to red blood cells (RBC). The characterization of the autoantigen in WAIHA has not yet led to definitive results, and the etiology of RBC autoantibodies remains unclear. An altered control of self-reactive IgG by autologous IgM has been proposed as the underlying mechanism of disease in WAIHA, suggesting that IgM–IgG immune complexes contribute to the pathophysiology of the disease. In the present study, we purified and characterized IgM from plasma of WAIHA patients and from healthy controls using FPLC-based protocols and optical biosensor technology, and investigated IgG present within the IgM fractions. We provide evidence that IgM–IgG immune complexes in plasma and associated with the RBC membrane are the characteristic feature of WAIHA, independent of the etiology of the disease. IgM–IgG immune complexes of WAIHA patients differ from IgM–IgG immune complexes of healthy individuals with regard to quantity and to structural composition. The data suggest that self-immunoglobulin is the original autoantigen underlying WAIHA. The molecular characterization of IgM–IgG immune complexes may define new targets for therapeutic intervention in WAIHA.

Alterations of self-reactive antibody repertoires in HIV disease: An insight into the role of T cells in the selection of autoreactive B cells

Infection with human immunodeficiency virus (HIV) is characterized by a progressive depletion of CD4 + T cells that parallels a dysfunction of the B cell compartment and a disturbed recognition of self-antigens. The relationship between T lymphocyte homeostasis and abnormalities in the selection of self-reactive B cells is not clear as yet. We have therefore compared repertoires of natural antibodies of healthy donors and of patients at various stages of HIV infection. The reactivity of IgM and IgG antibodies in plasma of healthy blood donors and of HIV-positive patients with high and low CD4 + T cell counts was assessed by semi-quantitative immunoblotting using self-antigens extracted from normal human tissues. Repertoires of reactivites were compared between groups of individuals by means of multiparametric statistical analysis. We observed that repertoires of self-reactive IgM and IgG from HIV-seropositive patients exhibited significantly altered patterns of reactivity, as compared to those of healthy controls. Further, self-reactive repertoires of IgM and IgG of patients with high CD4 + T cell counts differed significantly from those of patients with low CD4 + T cell counts. A longitudinal analysis of self-reactive antibody repertoires of progressor and non-progressor patients suggested an influence of CD4 + T cell counts on immunoglobulin reactivity toward self-antigens. These observations support the hypothesis that altered T cell/B cell interactions due to altered CD4 + T cell help severely impact on the selection of self-reactive antibody repertoires and may contribute to the onset of pathological autoimmunity in HIV disease.

Evolution of self-reactive IgG antibody repertoires in patients with relapsing-remitting multiple sclerosis

We have previously demonstrated a distortion of self-reactive IgG antibody repertoires in patients with multiple sclerosis (MS) compared to controls, by immunoblotting assays, using human brain homogenates. The analysis of the immune profiles against human brain antigens allowed us to distinguish MS patients, and to associate a particular pattern of reactivity for each clinical form of MS. The aim of the present study was to evaluate the evolution of such patterns in patients with relapsing-remitting MS (RRMS). In a first step, we confirmed, by western blotting using human brains as source of antigens, the existence of specific repertoires of IgG reactivity in whole serum collected from healthy subjects ( n = 32) and from untreated patients with RRMS ( n = 56). In a second step, the evaluation of patterns was performed at baseline and 1 year later in untreated RRMS patients ( n = 15), and in RRMS patients treated with IFN-β ( n = 41). In both groups, little change in IgG reactivity in whole serum was found. However, a higher degree of stability was noted in treated versus untreated patients ( P < 0.01). Our results have showed a specific and relatively stable pattern of reactivity for each RRMS individual tested against brain antigens even after a 1-year treatment prevailing in treated patients suggesting that IFN-β could stabilize IgG antibody repertoires.

Idiopathic membranous glomerulonephritis is associated with altered patterns of self-reactive IgM and IgG antibody repertoires.

Idiopathic membranous glomerulonephritis (MGN) is an immune complex nephropathy characterized by the subepithelial deposition of immunoglobulin (Ig)G. The pathogenesis of the disease remains largely unknown, but recent evidence suggests that human MGN may involve an autoimmune component. In the present study, we have analyzed the IgM and IgG antibody repertoires of patients with MGN towards self- and nonself-antigens using a technique of quantitative immunoblotting on a panel of whole human tissue or solubilized bacterial cell extracts as sources of antigens. Data were compared by means of multiparametric statistical analysis. We demonstrate that the antibody repertoires of self-reactive IgM and IgG in plasma of patients with MGN exhibit significantly altered patterns of reactivity, as compared with those of healthy controls. In contrast, multiparametric statistical analysis does not discriminate the reactivity patterns of IgM and IgG in plasma of patients and healthy controls towards nonself antigens. These observations indicate that a failure in the regulation of physiological self-reactivity is associated with immune complex nephropathy in MGN.

Altered Self-reactive Antibody Repertoires are a General Feature of Patients with Myelodysplastic Syndrome

Myelodysplastic syndrome (MDS) is characterized by an acquired clonal disorder of haematopoietic progenitor cells that results in inhibition of normal haematopoiesis and contributes to the development of haematological malignancies. Autoimmune syndromes may occur in MDS, but they are not a major clinical feature of the disease. In the present study, we have analysed the global antibody repertoires of IgM and IgG in plasma of 10 patients with MDS toward self- and non-self-antigens by quantitative immunoblotting. Myelodysplastic syndrome patients included in this study did not exhibit autoimmune symptoms nor secondary haematological neoplastic disease. Data were compared by means of multiparametric statistical analysis. We demonstrate that the antibody repertoires of self-reactive IgM and IgG of patients with MDS exhibit significantly altered patterns of reactivity, as compared to those of healthy individuals. In contrast, reactivity patterns of IgM in plasma of patients and of healthy controls toward non-self-antigens were similar, whereas reactivity patterns of IgG of patients and healthy subjects toward non-self-antigens were discriminated by multiparametric statistical analysis. These observations indicate that a broad disturbance of self-recognition mechanisms is a general feature of patients with MDS. A failure in the regulation of self-reactivity may contribute to the pathogenesis of MDS.

Altered control of self-reactive IgG by autologous IgM in patients with warm autoimmune hemolytic anemia

Warm autoimmune hemolytic anemia (WAIHA) is characterized by an accelerated clearance of red blood cells (RBCs) associated with the presence of anti-RBC immunoglobulin (Ig)G autoantibodies. In the present study, we analyzed the self-reactive IgG and IgM antibody repertoires of patients with WAIHA using a technique of quantitative immunoblotting on a panel of whole tissue extracts as sources of self-antigens. Data were compared by means of multiparametric statistical analysis. We demonstrate that self-reactive antibody repertoires of IgG purified from plasma and of IgG purified from RBC eluates do not differ between healthy donors and patients with WAIHA, whereas autoreactive repertoires of IgM from patients exhibit broadly altered patterns of reactivity as compared with those of healthy controls. We further demonstrate that IgG purified from eluates of RBCs of healthy donors induces agglutination of RBCs in an indirect Coombs assay to a similar extent as IgG purified from eluates of RBCs of patients with WAIHA. The capability of IgG to induce agglutination of RBCs is suppressed in unfractionated eluates of healthy donors' cells, whereas it is readily found in unfractionated eluates of patients' RBCs. IgM is an essential factor in controlling the ability of IgG in unfractionated RBC eluates to induce agglutination of RBCs. These observations indicate that anti-RBC IgG autoantibodies of patients with WAIHA share extensive similarity with natural antiRBC autoantibodies of healthy donors and suggest that defective control of IgG autoreactivity by autologous IgM is an underlying mechanism for autoimmune hemolysis in WAIHA. (Blood. 2000;95:328-335)