Fine Analysis of Lymphocyte Subpopulations in SARS-CoV-2 Infected Patients: Differential Profiling of Patients With Severe Outcome.

COVID-19 is caused by the human pathogen severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and has resulted in widespread morbidity and mortality. CD4+ T cells, CD8+ T cells and neutralizing antibodies all contribute to control SARS-CoV-2 infection. However, heterogeneity is a major factor in disease severity and in immune innate and adaptive responses to SARS-CoV-2. We performed a deep analysis by flow cytometry of lymphocyte populations of 125 hospitalized SARS-CoV-2 infected patients on the day of hospital admission. Five clusters of patients were identified using hierarchical classification on the basis of their immunophenotypic profile, with different mortality outcomes. Some characteristics were observed in all the clusters of patients, such as lymphopenia and an elevated level of effector CD8+CCR7- T cells. However, low levels of T cell activation are associated to a better disease outcome; on the other hand, profound CD8+ T-cell lymphopenia, a high level of CD4+ and CD8+ T-cell activation and a high level of CD8+ T-cell senescence are associated with a higher mortality outcome. Furthermore, a cluster of patient was characterized by high B-cell responses with an extremely high level of plasmablasts. Our study points out the prognostic value of lymphocyte parameters such as T-cell activation and senescence and strengthen the interest in treating the patients early in course of the disease with targeted immunomodulatory therapies based on the type of adaptive response of each patient.

Complement Alternative and Mannose-Binding Lectin Pathway Activation Is Associated With COVID-19 Mortality.

Background: The SARS-CoV-2 infection triggers excessive immune response resulting in increased levels of pro-inflammatory cytokines, endothelial injury, and intravascular coagulopathy. The complement system (CS) activation participates to this hyperinflammatory response. However, it is still unclear which activation pathways (classical, alternative, or lectin pathway) pilots the effector mechanisms that contribute to critical illness. To better understand the immune correlates of disease severity, we performed an analysis of CS activation pathways and components in samples collected from COVID-19 patients hospitalized in Grenoble Alpes University Hospital between 1 and 30 April 2020 and of their relationship with the clinical outcomes. Methods: We conducted a retrospective, single-center study cohort in 74 hospitalized patients with RT-PCR-proven COVID-19. The functional activities of classical, alternative, and mannose-binding lectin (MBL) pathways and the antigenic levels of the individual components C1q, C4, C3, C5, Factor B, and MBL were measured in patients' samples during hospital admission. Hierarchical clustering with the Ward method was performed in order to identify clusters of patients with similar characteristics of complement markers. Age was included in the model. Then, the clusters were compared with the patient clinical features: rate of intensive care unit (ICU) admission, corticoid treatment, oxygen requirement, and mortality. Results: Four clusters were identified according to complement parameters. Among them, two clusters revealed remarkable profiles: in one cluster (n = 15), patients exhibited activation of alternative and lectin pathways and low antigenic levels of MBL, C4, C3, Factor B, and C5 compared to all the other clusters; this cluster had the higher proportion of patients who died (27%) and required oxygen support (80%) or ICU care (53%). In contrast, the second cluster (n = 19) presented inflammatory profile with high classical pathway activity and antigenic levels of complement components; a low proportion of patients required ICU care (26%) and no patient died in this group. Conclusion: These findings argue in favor of prominent activation of the alternative and MBL complement pathways in severe COVID-19, but the spectrum of complement involvement seems to be heterogeneous requiring larger studies.

The Innate Part of the Adaptive Immune System.

The innate immune response provides a first line of defense against common microorganisms and, for more complex and/or recurring situations where pathogens must be eliminated, an adaptive immune response has emerged and evolved to provide better protection against subsequent infections. However, such dichotomy has to be reevaluated because innate B cells (e.g., B1 and marginal zone B cells) and the newly described innate lymphoid cells (iLC) have been found to exhibit innate-like properties, such as antigen internalization, regulatory B cell functions, and helper T cell activities. In addition, the production and function of natural antibodies (nAbs) by innate B cells and their capacity to activate the classical complement pathway constitute additional important mechanisms at the junction of innate and adaptive immunity as well as the recent integration of platelets into the innate immune spectrum. There is no doubt that these mechanisms present an advantage in immunity and homeostasis particularly during the first years of life, but arguments are arising to consider that these precursors may have detrimental effects in a variety of autoimmune/inflammatory diseases, allergies and cancers, as well as in response to immunotherapy. Accordingly, and as presented in this special issue of Clinical Reviews in Allergy and Immunology, a better comprehension of the key molecular and cellular actors implicated at the crossroads of the innate and adaptive immune response represents a new challenge in our understanding of the immunological and immunopathological responses.

The Immunopathology of Complement Proteins and Innate Immunity in Autoimmune Disease.

The complement is a powerful cascade of the innate immunity and also acts as a bridge between innate and acquired immune defence. Complement activation can occur via three distinct pathways, the classical, alternative and lectin pathways, each resulting in the common terminal pathway. Complement activation results in the release of a range of biologically active molecules that significantly contribute to immune surveillance and tissue homeostasis. Several soluble and membrane-bound regulatory proteins restrict complement activation in order to prevent complement-mediated autologous damage, consumption and exacerbated inflammation. The crucial role of complement in the host homeostasis is illustrated by association of both complement deficiency and overactivation with severe and life-threatening diseases. Autoantibodies targeting complement components have been described to alter expression and/or function of target protein resulting in a dysregulation of the delicate equilibrium between activation and inhibition of complement. The spectrum of diseases associated with complement autoantibodies depends on which complement protein and activation pathway are targeted, ranging from autoimmune disorders to kidney and vascular diseases. Nevertheless, these autoantibodies have been identified as differential biomarkers for diagnosis or follow-up of disease only in a small number of clinical conditions. For some autoantibodies, a clear relationship with clinical manifestations has been identified, such as anti-C1q, anti-Factor H, anti-C1 Inhibitor antibodies and C3 nephritic factor. For other autoantibodies, the origin and the functional consequences still remain to be elucidated, questioning about the pathophysiological significance of these autoantibodies, such as anti-mannose binding lectin, anti-Factor I, anti-Factor B and anti-C3b antibodies. The detection of autoantibodies targeting complement components is performed in specialized laboratories; however, there is no consensus on detection methods and standardization of the assays is a real challenge. This review summarizes the current panorama of autoantibodies targeting complement recognition proteins of the classical and lectin pathways, associated proteases, convertases, regulators and terminal components, with an emphasis on autoantibodies clearly involved in clinical conditions.

Flow cytometric analysis of neutrophil myeloperoxidase expression in peripheral blood for ruling out myelodysplastic syndromes: a diagnostic accuracy study.

Suspicion of myelodysplastic syndromes (MDS) is one of the commonest reasons for bone marrow aspirate in elderly patients presenting with persistent peripheral blood (PB) cytopenia of unclear etiology. A PB assay that accurately rules out MDS would have major benefits. The diagnostic accuracy of the intra-individual robust coefficient of variation (RCV) for neutrophil myeloperoxidase (MPO) expression measured by flow cytometric analysis in PB was evaluated in a retrospective derivation study (44 MDS cases and 44 controls) and a prospective validation study (68 consecutive patients with suspected MDS). Compared with controls, MDS cases had higher median RCV values for neutrophil MPO expression (40.2% vs 30.9%; P<0.001). The area under the receiver operating characteristic curve estimates were 0.94 [95% confidence interval (CI): 0.86-0.97] and 0.87 (95%CI: 0.76-0.94) in the derivation and validation studies, respectively. A RCV lower than 30% ruled out MDS with 100% sensitivity (95%CI: 78-100%) and 100% negative predictive value (95%CI: 83-100%) in the prospective validation study. Neutrophil MPO expression measured by flow cytometric analysis in PB might obviate the need for invasive bone marrow aspirate and biopsy for up to 29% of patients with suspected MDS.

Effects of immunoadsorption combined with membrane filtration on complement markers - results of a randomized, controlled, crossover study.

The complement system has been implicated in several kidney diseases, such as antibody-mediated rejection after kidney transplantation. Antibody-depletion techniques allow successful ABO- and/or HLA-incompatible transplantation. Considering the IgG removal, the use of semi-selective immunoadsorption (IA) has been advocated. However, because of results on incomplete IgM depletion, the adjunctive use of membrane filtration (MF) has been proposed to enhance the removal of macromolecules and to interfere with complement activation. This secondary endpoint analysis of a recently published randomized, controlled, cross-over trial was designed to investigate the effect of combined treatment IA + MF compared to IA alone on complement depletion. Two treatment sequences, a single session of IA + MF followed by IA (and vice versa), were analyzed with regard to C5b-9, properdin, and mannose-binding lectin (MBL) levels. Neither IA alone nor IA + MF provoked complement activation as demonstrated by stable low levels of C5b-9 after the procedure as compared to the previous. The combined treatment substantially lowered properdin (77% vs. 26% reduction, P < 0.0001) as well as MBL concentrations (81% vs. 11% reduction, P < 0.0001). Recovery of properdin and MBL levels appears to be longer after IA alone compared to IA + MF. Depletion of properdin and MBL levels may have potential clinical implications in the setting of kidney transplantation.

Diagnostic biologique des angioedèmes bradykiniques : les recommandations du CREAK.

Bradykinin mediated angioedema (BK-AE) can be associated either with C1Inhibitor deficiency (hereditary and acquired forms), either with normal C1Inh (hereditary form and drug induced AE as angiotensin converting enzyme inhibitors…). In case of high clinical suspicion of BK-AE, C1Inh exploration must be done at first: C1Inh function and antigenemy as well as C4 concentration. C1Inh deficiency is significant if the tests are below 50 % of the normal values and controlled a second time. In case of C1Inh deficiency, you have to identify hereditary from acquired forms. C1q and anti-C1Inh antibody tests are useful for acquired BK-AE. SERPING1 gene screening must be done if a hereditary angioedema is suspected, even if there is no family context (de novo mutation 15 %). If a hereditary BK-AE with normal C1Inh is suspected, F12 and PLG gene screening is suitable.

Accurate quantification of fourteen normal bone marrow cell subsets in infants to the elderly by flow cytometry.

BACKGROUND: Studies of normal bone marrow (BM) cell composition by flow cytometry are scarce. Presently, we aimed to quantify 14 cell subsets from infants to elderly patients. METHODS: Cell subsets in BM samples from 180 individuals without morphologically abnormal leukocytes were analyzed using a single combination of eight antibodies: CD3/CD10/CD38/CD19/CD36/CD16/CD34/CD45. RESULTS: By comparison with the Holdrinet score, we first validated the immature granulocyte/neutrophil (IGRA/N) ratio as a readily obtainable criterion of BM sample purity in 145 cases. Then, the 115 highly pure samples were selected (IGRA/N ≥ 1.2) and analyzed according to age group. CD34+ myeloblasts became progressively more infrequent with age: median 1.4% in infancy to 0.5% in the elderly. Neutrophils increased: 10.7% to 22.8%; all other myeloid subsets, IGRA, eosinophils, basophils and monocytes remained stable: respectively 40.3% to 46.7%, 2.0% to 2.8%, 0.2% to 0.3%, and 4.4% to 5.0% throughout life. Erythroblasts were lower in children (8.4% to 10.3%) than in adults (12.5% to 15.1%). For lymphoid cells, hematogones and transitional B-cells decreased: 15.5% to 0.6% and 3.6% to 0.1%, respectively; mature lymphocytes remained stable: B-cells: 1.4% to 2.8%, T-cells: 5.8% to 8.7%, and NK-cells: 0.7% to 1.4%. Plasma cells varied slightly: 0.1% to 0.5%. Differences of about 40% were seen in moderately pure (IGRA/N: 0.5 to 1.2) BM samples. CONCLUSION: We thus provide the first values for 14 myeloid and lymphoid subsets characterizing BM cell composition in 5 age ranges. They should provide important information when screening patients for hematological disorders or abnormal bone marrow development. © 2018 International Clinical Cytometry Society.

Antibodies targeting circulating protective molecules in lupus nephritis: Interest as serological biomarkers.

Lupus nephritis (LN) is one of the most frequent and severe manifestations of systemic lupus erythematosus (SLE), considered as the major predictor of poor prognosis. An early diagnosis of LN is a real challenge in the management of SLE and has an important implication in guiding treatments. In clinical practice, conventional parameters still lack sensitivity and specificity for detecting ongoing disease activity in lupus kidneys and early relapse of nephritis. LN is characterized by glomerular kidney injury, essentially due to deposition of immune complexes involving autoantibodies against cellular components and circulating proteins. One of the possible mechanisms of induction of autoantibodies in SLE is a defect in apoptotic cells clearance and subsequent release of intracellular autoantigens. Autoantibodies against soluble protective molecules involved in the uptake of dying cells, including complement proteins and pentraxins, have been described. In this review, we present the main autoantibodies found in LN, with a focus on the antibodies against these protective molecules. We also discuss their pathogenic role and conclude with their potential interest as serological biomarkers in LN.

Routinely used immunoassays do not detect circulating anti-GBM antibodies against native NC1 hexamer and EA epitope of the α3 chain of type IV collagen.

Detection of circulating anti-GBM antibodies has a key role for the diagnosis of Goodpasture syndrome but immunoassays using purified or recombinant alpha3(IV)NC1 as antigen do not recognize all anti-GBM antibodies. We show that anti-GBM antibodies directed against epitopes in their native conformation or cryptic epitopes are detected by indirect immunofluorescence.

Autoantibodies Targeting Ficolin-2 in Systemic Lupus Erythematosus Patients With Active Nephritis.

OBJECTIVE: Systemic lupus erythematosus (SLE) is a multisystem inflammatory disease characterized by the production of various autoantibodies. The aim of this study was to investigate the presence of anti-ficolin-2 antibodies in SLE patients and to evaluate the association between the levels of these autoantibodies, clinical manifestations, and disease activity. METHODS: This is a comparative study using a cohort of 165 SLE patients and 48 healthy subjects. SLE patients were further divided into 2 groups (low disease activity [SLE Disease Activity Index (SLEDAI) score ≤4, n = 88] and high disease activity [SLEDAI score >4, n = 77]). Clinical manifestations were defined according to the physician in charge. Active lupus nephritis (LN) was documented by kidney biopsy. Detection of anti-ficolin-2 antibodies was performed by enzyme-linked immunosorbent assay. RESULTS: Levels of anti-ficolin-2 autoantibodies were significantly higher in SLE patients as compared to healthy subjects and associated with SLEDAI score. They were found to be positive in 61 of 165 SLE patients (37%). The presence of anti-ficolin-2 antibodies was significantly related only to renal involvement, with a very high prevalence (86%) of anti-ficolin-2 antibodies in SLE patients with active LN. Patients with active proliferative LN had significantly more positive anti-ficolin-2 antibodies than those with nonproliferative LN. The combination of anti-ficolin-2, anti-ficolin-3, and anti-C1q demonstrated a very high specificity (98%) for the diagnosis of active LN. CONCLUSION: Our results support the usefulness of anti-ficolin-2 as a complementary serologic biomarker for the diagnosis of active lupus with renal manifestations.

One tube with eight antibodies for 14-part bone marrow leukocyte differential using flow cytometry.

BACKGROUND: Bone marrow analysis by flow cytometry is part of the routine diagnosis of hematological disorders in medical laboratories. Differential leukocyte count and identification of abnormal cell subsets is currently performed through morphological examination on bone marrow smears by skilled cytologists. In this work, we propose a single 8-color tube for providing equivalent information, using flow cytometry. METHODS: 99 bone marrow samples were classified into 2 groups, (i) 51 samples, obtained from either healthy donors (n = 4) or patients with various diseases at diagnosis or during remission that did not present a hematological malignancy (n = 47), and (ii) 48 pathological samples with quantitative and/or qualitative abnormalities. A panel of eight antibodies-CD3-FITC/CD10-PE/CD38-PerCP-Cy5.5/CD19-PECy7/CD36-APC/CD16-APC-H7/CD34-BV421/CD45-V500-was tested to identify the main cell subsets at different stages of maturation using a FACSCanto-II analyzer. RESULTS: We first proposed a strategy of sequential gating leading to the identification of 14 leukocyte subsets, that is, erythroblasts, monocytes, B-lymphoid cells from hematogones to plasma-cells (5 subsets), T- and NK-cells, polymorphonuclear cells (neutrophils, eosinophils, and basophils), myeloblasts and other immature granular cells. This approach was validated by comparing flow cytometry and microscopic morphological examination, both in cases of normal and abnormal samples. Interestingly, cell identification, and numeration by flow cytometry was easy to perform and highly reproducible. CONCLUSION: A very simple, rapid, and reproducible flow cytometric approach, using a combination of eight antibodies allows determination of the cellular composition of bone marrow with high precision. © 2016 International Clinical Cytometry Society.

Association between the Presence of Autoantibodies Targeting Ficolin-3 and Active Nephritis in Patients with Systemic Lupus Erythematosus.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by the production of multiple autoantibodies. Antibodies against Ficolin-3 were previously identified in the sera of some SLE patients, but their prevalence and significance have not been yet investigated. The aims of this study were to determine the prevalence of anti-ficolin-3 antibodies among SLE patients and to investigate their potential as diagnostic and/or prognostic biomarkers in SLE. In this retrospective study, sera from SLE patients (n = 165) were selected from a preexisting declared biological collection. Samples from healthy controls (n = 48) were matched with SLE sera. Disease activity was determined according to the SLEDAI score. Anti-ficolin-3, anti-dsDNA and anti-C1q antibodies levels were measured in sera by ELISA. First, a highly significant difference was found in the anti-ficolin-3 levels between SLE patients and healthy subjects. Anti-ficolin-3 antibodies were detected as positive in 56 of 165 (34%) SLE patients. The titer of anti-ficolin-3 antibodies was correlated with the SLEDAI score (r = 0.38, p<0.0001). The presence of anti-ficolin-3 antibodies was associated with anti-C1q and anti-dsDNA antibodies. Regarding associations with clinical manifestations, the presence of active lupus nephritis was significantly associated with the presence of anti-ficolin-3 antibodies (p≤0.001). This association with renal involvement was higher with anti-ficolin-3 or anti-C1q antibodies than with other auto-antibodies. Interestingly, the combination of anti-ficolin-3 and anti-C1q antibodies demonstrated higher specificity than any other serological biomarker. These results suggest that anti-ficolin-3 antibodies could be useful for the diagnosis of active nephritis in SLE patients.

Novel Strategy for Phenotypic Characterization of Human B Lymphocytes from Precursors to Effector Cells by Flow Cytometry.

A precise identification and phenotypic characterization of human B-cell subsets is of crucial importance in both basic research and medicine. In the literature, flow cytometry studies for the phenotypic characterization of B-lymphocytes are mainly focused on the description of a particular cell stage, or of specific cell stages observed in a single type of sample. In the present work, we propose a backbone of 6 antibodies (CD38, CD27, CD10, CD19, CD5 and CD45) and an efficient gating strategy to identify, in a single analysis tube, a large number of B-cell subsets covering the whole B-cell differentiation from precursors to memory and plasma cells. Furthermore, by adding two antibodies in an 8-color combination, our approach allows the analysis of the modulation of any cell surface marker of interest along B-cell differentiation. We thus developed a panel of seven 8-colour antibody combinations to phenotypically characterize B-cell subpopulations in bone marrow, peripheral blood, lymph node and cord blood samples. Beyond qualitative information provided by biparametric representations, we also quantified antigen expression on each of the identified B-cell subsets and we proposed a series of informative curves showing the modulation of seventeen cell surface markers along B-cell differentiation. Our approach by flow cytometry provides an efficient tool to obtain quantitative data on B-cell surface markers expression with a relative easy-to-handle technique that can be applied in routine explorations.

Unfolded protein response gene GADD34 is overexpressed in rheumatoid arthritis and related to the presence of circulating anti-citrullinated protein antibodies.

Growth arrest and DNA damage-inducible gene 34 (GADD34) is an inducible cofactor of protein phosphatase 1, which has an important role in the unfolded protein response. GADD34 has been shown to be necessary for type I interferon and proinflammatory cytokine production in response to viral infection in murine models. We investigate the expression of GADD34 in rheumatoid arthritis (RA), in which proinflammatory cytokines have an important pathogenic role. The objective of this study was to evaluate the potential of GADD34 expression as a biomarker in RA patients. We report a case-control study on GADD34 gene expression in peripheral blood mononuclear cells of patients (n = 75) with RA and age- and sex-matched healthy controls (n = 25). The study was approved by the relevant local ethics committees. GADD34 gene expression level in peripheral blood mononuclear cells was measured by quantitative PCR and analyzed with Mann-Whitney test. The relation between GADD34 gene overexpression and clinical or biological characteristics was analyzed with univariate and multivariate analysis. GADD34 gene expression was significantly higher in RA patients compared with healthy controls (p ≤ 0.001). Interestingly, GADD34 overexpression in PBMC of patients was related to the presence of circulating anti-citrullinated protein antibodies (p = 0.030). Data of this study strengthen the evidence of an unfolded protein response during the course of RA and provide an insight of the potential interest in GADD34 as a relevant marker for RA.

Ataxia with cerebellar lesions in mice expressing chimeric PrP-Dpl protein.

Mutations within the central region of prion protein (PrP) have been shown to be associated with severe neurotoxic activity similar to that observed with Dpl, a PrP-like protein. To further investigate this neurotoxic effect, we generated lines of transgenic (Tg) mice expressing three different chimeric PrP-Dpl proteins. Chi1 (amino acids 1-57 of Dpl replaced by amino acids 1-125 of PrP) and Chi2 (amino acids 1-66 of Dpl replaced by amino acids 1-134 of PrP) abrogated the pathogenicity of Dpl indicating that the presence of a N-terminal domain of PrP (23-134) reduced the toxicity of Dpl, as reported. However, when the amino acids 1-24 of Dpl were replaced by amino acids 1-124 of PrP, Chi3 Tg mice, which express the chimeric protein at a very low level, start developing ataxia at the age of 5-7 weeks. This phenotype was not counteracted by a single copy of full-length-PrP(c) but rather by its overexpression, indicating the strong toxicity of the chimeric protein Chi3. Chi3 Tg mice exhibit severe cerebellar atrophy with a significant loss of granule cells. We concluded that aa25 to aa57 of Dpl, which are not present in Chi1 and Chi2 constructs, confer toxicity to the protein. We tested this possibility by using the 25-57 Dpl peptide in primary culture of mouse embryo cortical neurons and found a significant neurotoxic effect. This finding identifies a protein domain that plays a role in mediating Dpl-related toxicity.

Prion replication in the hematopoietic compartment is not required for neuroinvasion in scrapie mouse model.

Fatal neurodegenerative prion diseases are caused by the transmissible PrP(Sc) prion agent whose initial replication after peripheral inoculation takes place in follicular dendritic cells present in germinal centers of lymphoid organs. However, prion replication also occurs in lymphoid cells. To assess the role of the hematopoietic compartment in neuroinvasion and prion replication, we generated chimeric mice, on a uniform congenic C57/BL6J background, by bone marrow replacement with hematopoietic cells expressing different levels of PrP protein. Nine different types of chimeric mice were inoculated intraperitoneally either with the lymphotropic Rocky Mountain Laboratory (RML) strain or the non lymphotropic ME-7 scrapie strain, at different doses. Here, we clearly demonstrate that overexpression of PrP by the hematopoietic system, or the lack of PrP expression by the bone marrow derived cells, does not change the incubation time period of the disease, even when the mice are infected at limiting doses. We conclude that the hematopoietic compartment is more or less permissive to prion replication, both for RML and ME-7, but does not play a role in neuroinvasion.

Sex effect in mouse and human prion disease.

Sex effect on the incubation period of variant Creutzfeldt-Jakob disease (vCJD) disease in human and ME-7 murine models was investigated. In the 167 vCJD cases reported in the United Kingdom as of January 2009, age at onset was significantly lower in female patients (by 2 years) than in male patients after stratification on birth cohort. In C57/Bl6N mice infected with ME-7 scrapie strain, incubation was shorter in female than in male mice. The incubation period increased in castrated male mice after intraperitoneal infection but not after intracerebral inoculation. In the absence of androgen receptors, the incubation period for prion disease increased after intraperitoneal inoculation. In ovariectomized or estrogen receptor alpha-defective female mice, no effect was observed on the incubation period of mouse prion disease. These results show that androgens influence the prion diseases incubation period after inoculation at a peripheral site.

Complement protein C1q forms a complex with cytotoxic prion protein oligomers.

A growing number of studies have investigated the interaction between C1q and PrP, but the oligomeric form of PrP involved in this interaction remains to be determined. Aggregation of recombinant full-length murine PrP in the presence of 100 mm NaCl allowed us to isolate three different types of oligomers by size-exclusion chromatography. In contrast to PrP monomers and fibrils, these oligomers activate the classical complement pathway, the smallest species containing 8-15 PrP protomers being the most efficient. We used Thioflavine T fluorescence to monitor PrP aggregation and showed that, when added to the reaction, C1q has a cooperative effect on PrP aggregation and leads to the formation of C1q-PrP complexes. In these complexes, C1q interacts through its globular domains preferentially with the smallest oligomers, as shown by electron microscopy, and retains the ability to activate the classical complement pathway. Using two cell lines, we also provide evidence that C1q inhibits the cytotoxicity induced by the smallest PrP oligomers. The cooperative interaction between C1q and PrP could represent an early step in the disease, where it prevents elimination of the prion seed, leading to further aggregation.