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The complement system plays a complex role in cancer. In clear cell renal cell carcinoma (ccRCC), local production of complement proteins drives tumor progression, but the mechanisms by which they do this are poorly understood. We found that complement activation, as reflected by high plasma C4d or as C4d deposits at the tumor site, was associated with poor prognosis in two cohorts of patients with ccRCC. High expression of the C4-activating enzyme C1s by tumor cells was associated with poor prognosis in three cohorts. Multivariate Cox analysis revealed that the prognostic value of C1s was independent from complement deposits, suggesting the possibility of complement cascade–unrelated, protumoral functions for C1s. Silencing of C1s in cancer cell lines resulted in decreased proliferation and viability of the cells and in increased activation of T cells in in vitro cocultures. Tumors expressing high levels of C1s showed high infiltration of macrophages and T cells. Modification of the tumor cell phenotype and T-cell activation were independent of extracellular C1s levels, suggesting that C1s was acting in an intracellular, noncanonical manner. In conclusion, our data point to C1s playing a dual role in promoting ccRCC progression by triggering complement activation and by modulating the tumor cell phenotype and tumor microenvironment in a complement cascade–independent, noncanonical manner. Overexpression of C1s by tumor cells could be a new escape mechanism to promote tumor progression.See related Spotlight by Magrini and Garlanda, p. 855.See article by Daugan et al., p. 909 (40).

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Purpose:Human telomerase reverse transcriptase (hTERT) is highly expressed in >85% of human tumors and is thus considered as a good tumor-associated antigen candidate for vaccine development. We conducted a phase I study to investigate the safety, tolerability, clinical response, and immunogenicity of INVAC-1, a DNA plasmid encoding a modified hTERT protein in patients with relapsed or refractory solid tumors.Patients and Methods:INVAC-1 was either administered by intradermal route followed by electroporation or by Tropis, a needle-free injection system. Safety and tolerability were monitored by clinical and laboratory assessments. Progression-free survival and overall survival were reported using Kaplan–Meier survival analysis. Immunogenicity was studied by ELISpot, Luminex, and Flow Cytometry.Results:Twenty-six patients were treated with INVAC-1 administered at three dose levels (100, 400, and 800 μg). Vaccination was well tolerated and no dose-limiting toxicity was reported. One treatment-related grade 3 SAE was reported. Fifty-eight percent of patients experienced disease stabilization. PFS was 2.7 months, median OS was 15 months, and 1-year survival was reached for 65% of patients. INVAC-1 vaccination stimulated specific anti-hTERT CD4 T-cell response as well as cytotoxic CD8 T-cell response. No evidence of peripheral vaccine-induced immunosuppression was observed.Conclusions:INVAC-1 vaccination was safe, well tolerated, and immunogenic when administered intradermally at the three tested doses in patients with relapsed or refractory cancers. Disease stabilization was observed for the majority of patients (58%) during the treatment period and beyond.See related commentary by Slingluff Jr, p. 529

Histological diagnosis, age, number of cycles received and Overall Survival

Immunophenotyping of circulating CD4 and CD8 T cells

Number and percentage of patients reporting studytreatment-related AEs per cycle

Clinical features of patients with OS 1 year

Flow Chart

Kaplan-Meier representation of Overall Survival and Progression Free Survival

This file compiles all the results identified as "data not shown" in the manuscript

Flow Cytometry antibody references

Best Overall Response

INTRODUCTION: Antiphospholipid antibodies (APA) clinical relevance in COVID-19 is controversial. We aimed to investigate the prevalence and prognostic value of conventional and non-conventional APA in COVID-19 patients. METHODS: This study was a multi-centric, prospective observational French cohort of patients hospitalized for COVID-19 suspicion. RESULTS: 249 patients were hospitalized for suspected COVID-19, including 154 with confirmed COVID-19 and 95 not confirmed. We found a significant increase in lupus anticoagulant (LA) positivity among COVID-19 positive patients (60.9% versus 23.7% in non-COVID19 patients, p

Objectives No reference data are available on repositories to measure precision of autoantibody assays. The scope of this study was to document inter- and intra-run variations of quantitative autoantibody assays based on a real-world large international data set. Methods Members of the European Autoimmunity Standardisation Initiative (EASI) group collected the data of intra- and inter-run variability obtained with assays quantifying 15 different autoantibodies in voluntary participating laboratories from their country. We analyzed the impact on the assay performances of the type of immunoassay, the number of measurements used to calculate the coefficient of variation (CVs), the nature and the autoantibody level of the internal quality control (IQC). Results Data were obtained from 64 laboratories from 15 European countries between February and October 2021. We analyzed 686 and 1,331 values of intra- and inter-run CVs, respectively. Both CVs were significantly dependent on: the method of immunoassay, the level of IQC with higher imprecision observed when the antibody levels were lower than 2-fold the threshold for positivity, and the nature of the IQC with commercial IQCs having lower CVs than patients-derived IQCs. Our analyses also show that the type of autoantibody has low impact on the assay’ performances and that 15 measurements are sufficient to establish reliable intra- and inter-run variations. Conclusions This study provides for the first time an international repository yielding values of intra- and inter-run variation for quantitative autoantibody assays. These data could be useful for ISO 15189 accreditation requirements and will allow clinical diagnostic laboratories to assure quality of patient results.

The ISO 15189 accreditation of biological analysis needs the validation of the analytical methods allowing the evaluation of their performance including all the factors that could influence the quality of their results. The field of autoimmunity includes many analyses and methods such as the indirect immunofluorescence technique (IIF) and the performance of this technique largely depends on the competency of staff members. For each staff member, the required levels of competency have to be precisely defined and evaluated after a period of formation before the final habilitation for the IIF technique. The French group of the international group called EASI (European autoimmunity standardisation initiative) proposes two habilitation forms to be filled with criteria, evidence and maintenance of target skills for the IIF preparation of slides and reading. These forms could be used as a model for the IIF formation and habilitation and have to be adapted to the routine practice of the laboratories.

Purpose: Human telomerase reverse transcriptase (hTERT) is highly expressed in >85% of human tumors and is thus considered as a good tumor-associated antigen candidate for vaccine development. We conducted a phase I study to investigate the safety, tolerability, clinical response, and immunogenicity of INVAC-1, a DNA plasmid encoding a modified hTERT protein in patients with relapsed or refractory solid tumors. Patients and Methods: INVAC-1 was either administered by intradermal route followed by electroporation or by Tropis, a needle-free injection system. Safety and tolerability were monitored by clinical and laboratory assessments. Progression-free survival and overall survival were reported using Kaplan–Meier survival analysis. Immunogenicity was studied by ELISpot, Luminex, and Flow Cytometry. Results: Twenty-six patients were treated with INVAC-1 administered at three dose levels (100, 400, and 800 μg). Vaccination was well tolerated and no dose-limiting toxicity was reported. One treatment-related grade 3 SAE was reported. Fifty-eight percent of patients experienced disease stabilization. PFS was 2.7 months, median OS was 15 months, and 1-year survival was reached for 65% of patients. INVAC-1 vaccination stimulated specific anti-hTERT CD4 T-cell response as well as cytotoxic CD8 T-cell response. No evidence of peripheral vaccine-induced immunosuppression was observed. Conclusions: INVAC-1 vaccination was safe, well tolerated, and immunogenic when administered intradermally at the three tested doses in patients with relapsed or refractory cancers. Disease stabilization was observed for the majority of patients (58%) during the treatment period and beyond. See related commentary by Slingluff Jr, p. 529

The interaction of some human antibodies with heme results in posttranslational acquisition of binding to various self- and pathogen-derived antigens. The previous studies on this phenomenon were performed with oxidized heme (Fe3+). In the present study, we elucidated the effect of other pathologically relevant species of heme, i.e., species that were formed after contact of heme with oxidizing agents such as hydrogen peroxide, situations in which heme’s iron could acquire higher oxidation states. Our data reveal that hyperoxidized species of heme have a superior capacity to heme (Fe3+) in triggering the autoreactivity of human IgG. Mechanistic studies demonstrated that oxidation status of iron was of critical importance for the heme’s effect on antibodies. We also demonstrated that hyperoxidized heme species interacted at higher affinities with IgG and that this binding occurred through a different mechanism as compared to heme (Fe3+). Regardless of their profound functional impact on the antigen-binding properties of antibodies, hyperoxidized species of heme did not affect Fc-mediated functions of IgG, such as binding to the neonatal Fc receptor. The obtained data contribute to a better understanding of the pathophysiological mechanism of hemolytic diseases and of the origin of elevated antibody autoreactivity in patients with some hemolytic disorders.

SARS-CoV-2 infection leads to a highly variable clinical evolution, ranging from asymptomatic to severe disease with acute respiratory distress syndrome, requiring intensive care units (ICU) admission. The optimal management of hospitalized patients has become a worldwide concern and identification of immune biomarkers predictive of the clinical outcome for hospitalized patients remains a major challenge. Immunophenotyping and transcriptomic analysis of hospitalized COVID-19 patients at admission allow identifying the two categories of patients. Inflammation, high neutrophil activation, dysfunctional monocytic response and a strongly impaired adaptive immune response was observed in patients who will experience the more severe form of the disease. This observation was validated in an independent cohort of patients. Using in silico analysis on drug signature database, we identify differential therapeutics that specifically correspond to each group of patients. From this signature, we propose a score—the SARS-Score—composed of easily quantifiable biomarkers, to classify hospitalized patients upon arrival to adapt treatment according to their immune profile.

Hemolytic uremic syndrome (HUS), a thrombotic microangiopathy (TMA) with kidney involvement, is a rare condition in patients with monoclonal gammopathy. In the absence of known causes of TMA, the role of complement activation in endothelial injury in patients with monoclonal gammopathy remains unknown and was the focus of this investigation.Case series.We studied the 24 patients in the French national registry of HUS between 2000 and 2020 who had monoclonal gammopathy without other causes of secondary TMA. We provide the clinical histories and complement studies of these patients.Monoclonal gammopathy-associated TMA with kidney involvement is estimated to be 10 times less frequent than adult atypical HUS (aHUS) in the French national registry. It is characterized by severe clinical features, with 17 of 24 patients requiring dialysis at disease onset, and with median renal survival of only 20 months. TMA-mediated extrarenal manifestations, particularly cutaneous and neurological involvement, were common and associated with poor overall prognosis. Complement studies identified low C3, normal C4, and high soluble C5b-9 levels in 33%, 100%, and 77% of tested patients, respectively, indicating a contribution of the alternative and terminal complement pathways in the pathophysiology of the disease. Genetic abnormalities in complement genes known to be associated with aHUS were found in only 3 of 17 (17%) who were tested.Retrospective study without comparison group; limited number of patients, limited available blood samples.Within the spectrum of TMA, TMA associated with monoclonal gammopathy represents a distinct subset. Our findings suggest that HUS associated with monoclonal immunoglobulin is a complement-mediated disease akin to aHUS.

Hemolytic uremic syndrome (HUS) is a leading cause of acute kidney injury in children. Although international guidelines emphasize comprehensive evaluation and treatment with eculizumab, access to diagnostic and therapeutic facilities is limited in most developing countries. The burden of Shiga toxin-associated HUS in India is unclear; school-going children show high prevalence of anti-factor H (FH) antibodies. The aim of the consensus meeting was to formulate guidelines for the diagnosis and management of HUS in children, specific to the needs of the country. Four workgroups performed literature review and graded research studies addressing (i) investigations, biopsy, genetics, and differential diagnosis; (ii) Shiga toxin, pneumococcal, and infection-associated HUS; (iii) atypical HUS; and (iv) complement blockade. Consensus statements developed by the workgroups were discussed during a consensus meeting in March 2017. An algorithm for classification and evaluation was developed. The management of Shiga toxin-associated HUS is supportive; prompt plasma exchanges (PEX) is the chief therapy in patients with atypical HUS. Experts recommend that patients with anti-FH-associated HUS be managed with a combination of PEX and immunosuppressive medications. Indications for eculizumab include incomplete remission with plasma therapy, life-threatening features, complications of PEX or vascular access, inherited defects in complement regulation, and recurrence of HUS in allografts. Priorities for capacity building in regional and national laboratories are highlighted. Limited diagnostic capabilities and lack of access to eculizumab prevent the implementation of international guidelines for HUS in most developing countries. We propose practice guidelines for India, which will perhaps be applicable to other developing countries.

2523 Background: Immune checkpoint inhibitors (ICIs), used alone or as a combination are standard of care in many cancers. Generally well tolerated, they can generate immune-related adverse events (irAEs). No biomarkers are available to identify patients who are more likely to develop irAEs. The aim of this study was to assess the association between preexisting autoantibodies, occurrence of irAEs and survival outcomes. Methods: We performed a prospective study including 221 patients receiving ICIs for advanced solid tumors between May 2015 and July 2021. Autoantibodies testing (anti-neutrophil cytoplasmic, anti-nuclear, thyroid peroxidase and thyroglobulin) was performed before ICIs initiation. The associations among preexisting autoantibodies, the occurrence, the severity, the delay of irAEs and the survival outcomes, including progression-free survival (PFS) and overall survival (OS) were analyzed. Statistical analyses were performed with T-test, Cox regression models, univariate and multivariate analyses and Kaplan-Meier’s method. Results: Of the 221 patients, 151 (68%) were men, the median age was 66,5 (range 21-90) years and 103 (81%) had an ECOG-PS of 0 or 1. Seventy-three percent (n=162) received an anti-PD-(L)1 in monotherapy, 27% (n=59) an anti-PD-(L)1 in combination, for a renal cell carcinoma in 45% (n=99) and a lung carcinoma in 41% (n=90). In total, 129 (58%) patients had preexisting antibodies. IrAEs were significantly more frequent in patients with preexisting autoantibodies: 64 patients (50%) in the positive group vs. 20 patients (22%) in the negative group, OR = 3.5 (95%CI=1.8 - 6.8), p=0.00002. Median time interval between ICI initiation and irAE was shorter in the positive group vs. the negative group, 13 weeks (IQR=43weeks) vs. 28.5 weeks (IQR=12weeks) respectively (p=0.01). Twelve patients (9.4%) experienced multi toxicities in the positive group vs. two (2%) in the negative group, OR=4.5 (95%CI0.98-36), p=0.04. ICIs exposure was identical in preexisting and non-preexisting autoantibodies groups. After a median follow-up of 25 months (95%CI=19-31), median PFS and OS were significantly longer among patients experiencing irAE: 12.6 months (95%CI=11-22.7) vs 5 months (95%CI=4.2-7.0), p= 0.0003 and 30 months (95%CI= 22.7-NR) vs 21 months (95%CI=15-34.6), p= 0.016. In multivariate analyses irAEs remain statistically associated with survival outcomes. Preexisting autoantibodies were not associated with survival outcomes. Conclusions: The presence of preexisting autoantibodies is significantly associated with the occurrence of irAE in patients treated with ICIs. Earlier and multiple irAEs were observed in the presence of preexisting autoantibodies. Thus, these biomarkers could help to identify patients at risk of irAEs and would prompt us to closely monitor them.

The complement system plays a complex role in cancer. In clear cell renal cell carcinoma (ccRCC), local production of complement proteins drives tumor progression, but the mechanisms by which they do this are poorly understood. We found that complement activation, as reflected by high plasma C4d or as C4d deposits at the tumor site, was associated with poor prognosis in two cohorts of patients with ccRCC. High expression of the C4-activating enzyme C1s by tumor cells was associated with poor prognosis in three cohorts. Multivariate Cox analysis revealed that the prognostic value of C1s was independent from complement deposits, suggesting the possibility of complement cascade–unrelated, protumoral functions for C1s. Silencing of C1s in cancer cell lines resulted in decreased proliferation and viability of the cells and in increased activation of T cells in in vitro cocultures. Tumors expressing high levels of C1s showed high infiltration of macrophages and T cells. Modification of the tumor cell phenotype and T-cell activation were independent of extracellular C1s levels, suggesting that C1s was acting in an intracellular, noncanonical manner. In conclusion, our data point to C1s playing a dual role in promoting ccRCC progression by triggering complement activation and by modulating the tumor cell phenotype and tumor microenvironment in a complement cascade–independent, noncanonical manner. Overexpression of C1s by tumor cells could be a new escape mechanism to promote tumor progression. See related Spotlight by Magrini and Garlanda, p. 855. See article by Daugan et al., p. 909 (40).

SARS-CoV-2 infection leads to a highly variable clinical evolution, ranging from asymptomatic to severe disease with acute respiratory distress syndrome, requiring intensive care units (ICU) admission. The optimal management of hospitalized patients has become a worldwide concern and identification of immune biomarkers predictive of the clinical outcome for hospitalized patients remains a major challenge. Immunophenotyping and transcriptomic analysis of hospitalized COVID-19 patients at admission allow identifying the two categories of patients. Inflammation, high neutrophil activation, dysfunctional monocytic response and a strongly impaired adaptive immune response was observed in patients who will experience the more severe form of the disease. This observation was validated in an independent cohort of patients. Using in silico analysis on drug signature database, we identify differential therapeutics that specifically correspond to each group of patients. From this signature, we propose a score—the SARS-Score—composed of easily quantifiable biomarkers, to classify hospitalized patients upon arrival to adapt treatment according to their immune profile.

Impairment of the complement regulatory protein Factor H (FH) is implicated in the physiopathological mechanisms of different diseases like atypical hemolytic and uremic syndrome and C3 glomerulopathies. It may be due to genetic abnormalities or acquired with the development of autoantibodies. FH has several ligands; therefore, the exploration of its functions requires to perform different tests. Among them, two hemolytic tests are very useful because they give specific and complementary information about FH functions. The first one is dedicated to explore the FH capacity to dissociate the alternative pathway C3 convertase, whereas the second one is designed to explore the capacity of FH to bind cell surfaces and to protect them from complement attack. This chapter describes the procedures to perform these two hemolytic tests, exploring in a complementary way the FH functionality.

Sheep erythrocytes (SE) are commonly used in complement functional tests. Non sensitized SE are useful to study the FH activity of cell protection. Indeed, as the cell surface of sheep erythrocytes is rich in sialic acids, Factor H (FH) is able to bind on it and therefore they represent a model of nonactivating surface. Because of their high capacity of complement regulation SE need to be modified to explore other functionality of the complement pathways, like the Complement hemolytic 50 (CH50) or the AP C3 convertase decay assays. For these tests, SE are sensitized with an anti-sheep red blood cell stroma antibody. In presence of serum or plasma complement components, sensitized SE may initiate complement cascade activation via the classic pathway explored in the CH50 assay. Sensitized SE may also be used to prepare C3b-coated SE that, with the use of buffers favoring AP, are suitable for the C3 Nef hemolytic assay and for the hemolytic assay studying the AP decay activity of FH. In this chapter we describe how to prepare SE for these different hemolytic tests.

Sheep erythrocytes (SE) are commonly used in complement functional tests. Non sensitized SE are useful to study the FH activity of cell protection. Indeed, as the cell surface of sheep erythrocytes is rich in sialic acids, Factor H (FH) is able to bind on it and therefore they represent a model of nonactivating surface. Because of their high capacity of complement regulation SE need to be modified to explore other functionality of the complement pathways, like the Complement hemolytic 50 (CH50) or the AP C3 convertase decay assays. For these tests, SE are sensitized with an anti-sheep red blood cell stroma antibody. In presence of serum or plasma complement components, sensitized SE may initiate complement cascade activation via the classic pathway explored in the CH50 assay. Sensitized SE may also be used to prepare C3b-coated SE that, with the use of buffers favoring AP, are suitable for the C3 Nef hemolytic assay and for the hemolytic assay studying the AP decay activity of FH. In this chapter we describe how to prepare SE for these different hemolytic tests.

Impairment of the complement regulatory protein Factor H (FH) is implicated in the physiopathological mechanisms of different diseases like atypical hemolytic and uremic syndrome and C3 glomerulopathies. It may be due to genetic abnormalities or acquired with the development of autoantibodies. FH has several ligands; therefore, the exploration of its functions requires to perform different tests. Among them, two hemolytic tests are very useful because they give specific and complementary information about FH functions. The first one is dedicated to explore the FH capacity to dissociate the alternative pathway C3 convertase, whereas the second one is designed to explore the capacity of FH to bind cell surfaces and to protect them from complement attack. This chapter describes the procedures to perform these two hemolytic tests, exploring in a complementary way the FH functionality.

BackgroundNatural killer (NK) cells play a crucial role in tumor immunosurveillance through their cytotoxic effector functions and their capacity to interact with other immune cells to build a coordinated antitumor immune response. Emerging data reveal NK cell dysfunction within the tumor microenvironment (TME) through checkpoint inhibitory molecules associated with a regulatory phenotype.ObjectiveWe aimed at analyzing the gene expression profile of intratumoral NK cells compared with non-tumorous NK cells, and to characterize their inhibitory function in the TME.MethodsNK cells were sorted from human lung tumor tissue and compared with non- tumoral distant lungs.ResultsIn the current study, we identify a unique gene signature of NK cell dysfunction in human non-small cell lung carcinoma (NSCLC). First, transcriptomic analysis reveals significant changes related to migratory pattern with a downregulation of sphingosine-1-phosphate receptor 1 (S1PR1) and CX3C chemokine receptor 1 (CX3CR1) and overexpression of C-X-C chemokine receptor type 5 (CXCR5) and C-X-C chemokine receptor type 6 (CXCR6). Second, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and killer cell lectin like receptor (KLRC1) inhibitory molecules were increased in intratumoral NK cells, and CTLA-4 blockade could partially restore MHC class II level on dendritic cell (DC) that was impaired during the DCs/NK cell cross talk. Finally, NK cell density impacts the positive prognostic value of CD8+ T cells in NSCLC.ConclusionsThese findings demonstrate novel molecular cues associated with NK cell inhibitory functions in NSCLC.

Background: ISO 15189 accreditation remains a challenge for specialized laboratories. In the field of autoimmunity, beside the crucial problem of absence of standardization, laboratories have to manage the analytical performances of the large panel of assays in terms of sensitivity and specificity, but also on their measurement precision for which no reference values are available on biorepositories.Methods: As an initiative of the French EASI (European Autoimmunity Standardization Initiative) group, French clinical diagnostic laboratories were requested to participate in a survey aiming to analyze the coefficients of variation (CVs) of intra-run and inter-run variability obtained with assays quantifying 14 different autoantibodies. Two performance goals corresponding to the 90th percentile and the 50th percentile (lowest CV values reached by 90% and 50% of laboratories respectively) defined for three levels of concentration were calculated. The impact on the assay performances of the number of measurements, of the nature of the internal quality control (IQC) and the type of immunoassay, was also analyzed.Results: 414 and 616 values of intra-run and inter-run CVs were collected, respectively. The 50th percentile performance goals were comprised between 1.0% and 8.9% for the intra-run CVs, and between 1.8% and 14.6% for the inter-run CVs. At 90th percentile, the performance goals were comprised between 3.2% and 13.5% for the intra-run CVs, and between 7.3% and 30.8% for the inter-run CVs. CVs calculated from 10 values were similar to those obtained from more values. Higher imprecision was observed when the antibody levels of the IQC was lower than 2 fold the positive threshold. Commercial IQCs gave lower CVs than IQCs derived from patient samples.Conclusion: Our results allow proposing some acceptability limits for the precision performances of the autoantibody assays, compatible with the reality of life in diagnostic laboratories and clinical care.

The complement system is a powerful and druggable innate immune component of the tumor microenvironment. Nevertheless, it is challenging to elucidate the exact mechanisms by which complement affects tumor growth. In this study, we examined the processes by which the master complement regulator factor H (FH) affects clear cell renal cell carcinoma (ccRCC) and lung cancer, two cancers in which complement overactivation predicts poor prognosis. FH was present in two distinct cellular compartments: the membranous (mb-FH) and intracellular (int-FH) compartments. Int-FH resided in lysosomes and colocalized with C3. In ccRCC and lung adenocarcinoma, FH exerted protumoral action through an intracellular, noncanonical mechanism. FH silencing in ccRCC cell lines resulted in decreased proliferation, due to cell-cycle arrest and increased mortality, and this was associated with increased p53 phosphorylation and NFκB translocation to the nucleus. Moreover, the migration of the FH-silenced cells was reduced, likely due to altered morphology. These effects were cell type–specific because no modifications occurred upon CFH silencing in other FH-expressing cells tested: tubular cells (from which ccRCC originates), endothelial cells (human umbilical vein endothelial cells), and squamous cell lung cancer cells. Consistent with this, in ccRCC and lung adenocarcinoma, but not in lung squamous cell carcinoma, int-FH conferred poor prognosis in patient cohorts. Mb-FH performed its canonical function of complement regulation but had no impact on tumor cell phenotype or patient survival. The discovery of intracellular functions for FH redefines the role of the protein in tumor progression and its use as a prognostic biomarker or potential therapeutic target. See article by Daugan et al., p. 891 (36).

Special conditions associated with laboratory autoimmune testing are not well compatible with recent developments in regulatory frameworks such as EN/ISO 15189 accreditation or in vitro diagnostic medical device regulation (IVD-R). In addition, international recommendations, guidelines and disease criteria are poorly defined with respect to requirements on autoantibody testing. Laboratory specialists from Austria, Belgium, Croatia, Estonia, Finland, France, Germany, Greece, Hungary, Italy, Norway, Poland, Portugal, South Africa, Spain, Sweden, Switzerland, and The Netherlands collected information, reported national experience, and identified quality issues in relation to autoantibody testing that require consensus on interpretation of the regulatory frameworks and guidelines. This process has been organized by the European Autoimmunity Standardisation Initiative (EASI). By identifying the critical items and looking for a consensus, our objective was to define a framework for, in particular, EN/ISO accreditation purposes. Here, we present a review of current publications and guidelines in this field to unify national guidelines and deliver in this way a European handout on quality control and accreditation for laboratories involved in autoantibody testing. We focus on quality items that can be checked during accreditation visits. Despite various local varieties, we encountered an overwhelming dedication to quality assurance in all contributing countries. ispartof: AUTOIMMUNITY HIGHLIGHTS vol:11 issue:1 ispartof: location:England status: published

Background INVAC-1 is an optimized DNA plasmid encoding an inactive form of human Telomerase Reverse Transcriptase (hTERT), a universal tumor antigen expressed in most of human tumors with little or no expression in normal somatic cells. Primary pharmacodynamics, safety and toxicology studies showed that INVAC-1 was enzymatically inactive, immunogenically safe and well tolerated. In murine models, we demonstrated that INVAC-1 was able to induce hTERT specific cellular immune responses with CD4+ Th1 effector and memory CD8+ T-cells as well as slow tumor growth and increase survival rate by 50% in tumor-bearing mice. Methods We conducted a First-In-Human (FIH) study, 2-centre, Phase I, open label, 3+3 escalation design and multiple dose study examining the safety and tolerability of INVAC-1 administered at three dose levels (100, 400 and 800 µg) in 26 patients with relapsed or solid refractory tumors. INVAC-1 was administered either by intradermal (ID) injection followed by electroporation (EP) (n=20) or by Tropis® Needle Free Injection System (n=6). Results INVAC-1 vaccination was safe and well tolerated when administered ID (either with EP or by Tropis®) at the three tested doses. Only one treatment-related grade 3 SAE was reported. 58% of patients experienced disease stabilization up to 9.9 months. One-year survival was reached for 65% of patients. INVAC-1 elicited both hTERT specific Th1-dominant CD4 and cytotoxic CD8 T cell responses with no vaccine-induced peripheral immunosuppression. Anti-hTERT immune responses were enhanced by adding anti-PD-1 immune checkpoint inhibitor ex vivo. In addition, INVAC-1 vaccination was able to promote epitope spreading. Finally, correlation analysis between clinical and immunological data showed that patients with OS >1 year presented a significantly higher hTERT immune response after INVAC-1 vaccination compared to patients with OS Citation Format: Julie Garibal, Jacques Medioni, Luis Teixeira, Olivier Adotevi, Marie-Agnès Dragon-Durey, Caroline Laheurte, Claire Germain, Marie Escande, Maria Wehbe, Valérie Doppler, Thierry Huet, Pierre Langlade Demoyen. INVAC-1, an optimized telomerase DNA vaccine in patients with advanced solid tumors: Final results of first-in-human phase I study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr CT021.

Anti-neutrophil cytoplasmic antibodies (ANCA) are mainly searched for the diagnosis of autoimmune vasculitis. They may be found also in other conditions with chronic inflammation. Testing ANCA is based on two main technics: indirect immunofluorescence (IFI) and immunochemical technics to identify the antigenic specificity of the autoantibodies. There is heterogeneity among the laboratories' daily practice. An international group called EASI (European autoimmunity standardisation initiative), composed of 15 countries, comprising France, works to harmonize the practices of the biological diagnosis of the autoimmune diseases. It elaborated a survey consisting of 54 questions related to the analytic parameters of the technics, the algorithms for their use and their biological interpretation; and submitted it to European laboratories. We propose an analysis of the answers obtained from 36 French laboratories specialized in autoimmunity. We compare them to the ones obtained from the other countries and discussed them according to the international recommendations. The analysis reveals a predominant use of IFI as a first step with variable strategies for the identification of the antigenic specificity of the autoantibodies. Overall, the practices are chiefly conformed to the recommendations for the diagnosis of vasculitis, but they are less consensual when the ANCA are performed in other clinical situations.

Hypertension is common in hemolytic uremic syndrome (HUS) and often difficult to control. Local renin-angiotensin activation is believed to be an important part of thrombotic microangiopathy, leading to a vicious cycle of progressive renal injury and intractable hypertension. This has been demonstrated in vitro via enhanced tissue factor expression on glomerular endothelial cells which is enhanced by angiotensin II. We report two pediatric cases of atypical HUS with severe refractory malignant hypertension, in which we targeted the renin-angiotensin system by using intravenous (IV) enalaprilat, oral aliskiren, and oral enalapril with quick and dramatic response of blood pressure. Both drugs, aliskiren and IV enalaprilat, were effective in controlling hypertension refractory to multiple antihypertensive medications. These appear to be promising alternatives in the treatment of severe atypical HUS-induced hypertension and hypertensive emergency.

Much progress has been made in understanding the pathophysiology and treatment of atypical hemolytic uremic syndrome (aHUS). Plasma therapy is the mainstay of treatment for aHUS. The availability of the first effective anti-complement therapeutic agent, eculizumab, has dramatically changed the outlook of this disease. However, its use in clinical practice raises important questions, such as who should receive the drug, when to start such therapy, and is it safe to stop treatment once the disease is controlled. We describe here for the 1st time in India, use of eculizumab in a 12-year-old boy with aHUS. We also describe in this report challenges faced in procuring the drug, and an ideal, evidence-based method of treating aHUS in children.

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.

The innate immune complement system is a powerful defense cascade against pathogens, but can induce host tissue damage when overactivated. In pathological conditions, mainly but not restricted to renal diseases, such as lupus nephritis, atypical hemolytic uremic syndrome, and C3 glomerulopathies, complement is overactivated or dysregulated by autoantibodies directed against its components and regulators. Among the key autoantibody targets are the initiator of the classical complement pathway C1q, the alternative pathway regulator Factor H, the components of the alternative pathway C3 convertase complex C3 and Factor B and the convertase complex itself. This methodological article describes our experience with a method for detection of anti-complement autoantibodies in real time using surface plasmon resonance-based technology. It allows label-free evaluation of the binding efficacy and stability of the formed antigen-antibody complexes.

The complement system is a part of the immune system implicated in host defense against pathogens and damaged cells and Factor H is the main regulatory protein of this powerful enzymatic cascade. Autoantibodies directed against Factor H (anti-FH antibodies) are implicated in different pathologies mainly atypical hemolytic and uremic syndrome and C3 glomerulopathies. The detection and quantification of these autoantibodies are crucial for the clinical management of the patients.Anti-Factor H antibodies are detected and quantified by an ELISA assay. The aim of this chapter is to describe the procedure to determine anti-FH autoantibodies and to provide information about their biological significance.

C3 glomerulopathy (C3G) results from acquired or genetic abnormalities in the complement alternative pathway (AP). C3G with monoclonal immunoglobulin (MIg-C3G) was recently included in the spectrum of “monoclonal gammopathy of renal significance.” However, mechanisms of complement dysregulation in MIg-C3G are not described and the pathogenic effect of the monoclonal immunoglobulin is not understood. The purpose of this study was to investigate the mechanisms of complement dysregulation in a cohort of 41 patients with MIg-C3G. Low C3 level and elevated sC5b-9, both biomarkers of C3 and C5 convertase activation, were present in 44 and 78% of patients, respectively. Rare pathogenic variants were identified in 2/28 (7%) tested patients suggesting that the disease is acquired in a large majority of patients. Anti-complement auto-antibodies were found in 20/41 (49%) patients, including anti-FH (17%), anti-CR1 (27%), anti-FI (5%) auto-antibodies, and C3 Nephritic Factor (7%) and were polyclonal in 77% of patients. Using cofactor assay, the regulation of the AP was altered in presence of purified IgG from 3/9 and 4/7 patients with anti-FH or anti-CR1 antibodies respectively. By using fluid and solid phase AP activation, we showed that total purified IgG of 22/34 (65%) MIg-C3G patients were able to enhance C3 convertase activity. In five documented cases, we showed that the C3 convertase enhancement was mostly due to the monoclonal immunoglobulin, thus paving the way for a new mechanism of complement dysregulation in C3G. All together the results highlight the contribution of both polyclonal and monoclonal Ig in MIg-C3G. They provide direct insights to treatment approaches and opened up a potential way to a personalized therapeutic strategy based on chemotherapy adapted to the B cell clone or immunosuppressive therapy.

The detection and the quantification of specific antibodies represent essential tools for the diagnosis and for the biological monitoring of immune humoral response in many clinical situations in particular in autoimmune diseases or in the context of immunotherapy using monoclonal antibodies. This article focuses on the development of a specific antibody measuring method (Patent n°PCT/IB2014/064437). The principle of this method is based on the combined use of a monoclonal antibody as standard and the protein G as immunoglobulins detecting agent. We performed a complete analytical validation of this method for the quantification of antibodies in three different applications: autoantibodies, alloantibodies and therapeutic monoclonal antibody. The results showed good performances compatible with the use of these assays as diagnostic tools. This method allows avoiding the use of products from human origin as reagent that causes ethical and infectious concerns but also storage and long term stock management problems. Moreover, this approach is particularly useful when no commercial reagent is available, especially in the case of rare diseases.

International audience; BACKGROUND: Prodromal symptoms are frequently reported in the atypical form of Hemolytic uremic syndrome (aHUS) suggesting implication of infectious triggers. Some pathogens may also play a role in the mechanisms of production of autoantibody directed against Factor H (FH), a complement regulator, leading to aHUS.METHODS: The presence of 15 gastrointestinal (GI) pathogens was investigated by using xTAG-based multiplex PCR techniques on stools collected at the acute phase in a cohort of Indian HUS children classified according to the presence or absence of anti-FH autoantibodies.RESULTS: Prevalence of pathogens in patients with anti-FH antibody (62.5%) was twice that in those without (31.5%). Different pathogens were detected, the most frequent being Clostridium difficile, Giardia intestinalis, Salmonella, Shigella, Rotavirus, Norovirus and Entamoeba histolytica. No stool was positive for Shigatoxin.CONCLUSION: This study reveals a higher prevalence of GI pathogens in anti-FH positive than in negative patients. No single pathogen was implicated exclusively in one form of HUS. These pathogens may play a role in the disease initiation by inducing complement activation or an autoimmune response.