成人麻疹细胞免疫与CD4+ CD25+调节性T细胞的关系

目的 了解CD4+CD25+调节性T细胞(Treg)与麻疹患者细胞免疫的关系.方法 采集34例成年麻疹患者和27例健康对照的外周血,采用流式细胞术进行CD4+CD25+细胞和FoxP3+细胞的检测.用免疫磁珠从外周血单个核细胞(PBMC)分选出CD4+CD25+T细胞和CD4+CD25-T细胞,分别用抗-CD3单克隆抗体、BCG和NV减毒株进行刺激培养,收集培养上清,用ELISA法检测培养上清中的IFN-γ和IL-10.结果 麻疹患者外周血白细胞、淋巴细胞及CD3+CC4·细胞比例明显降低,而CD4+CD25+细胞与FoxP3+细胞的比例则与对照无显著差别;CD4+CD25+细胞明显抑制抗CD3刺激下CD4+CD25-T细胞产生IFN-γ,且患者和对照没有差别,而IL-10的产生没有这种改变.结论 成人麻疹患者的的免疫抑制与Treg的抑制作用无关. 4+CD25+T细胞和CD4+CD25-T细胞,分别用抗-CD3单克隆抗体、BCG和NV减毒株进行刺激培养,收集培养上清,用ELISA法检测培养上清中的IFN-γ和IL-10.结果 麻疹患者外周血白细胞、淋巴细胞及CD3+CC4+细胞比例明显降低,而CD4+CD2 +细胞与FoxP3+细胞的比例则与对照无显著差别;CD4+CD25+细胞明显抑制抗CD3刺激下CD4+CD25-T细胞产生IFN-γ,且患者和对照没有差别,而IL-10的产生     

T-Cell stimulation and regulation: With complements from CD46

Crosslinking of CD46 and CD3 on naïve human CD4⁺ T-lymphocytes induces interleukin-10 secretion and granzyme B expression. These highly proliferative T-regulatory type 1-like T-regulatory T-cells (Tregs) can suppress an immune response. We propose that this process is important in the prevention of chronic inflammation such as at epithelial borders and in deactivation of a successful immune response. Relative to the latter, once a complement-fixing polyclonal antibody response has been mounted, in most cases, the pathogen will be rapidly destroyed. At this time, the C3b/C4b-bearing immune complexes could initiate the deactivation arm of an immune response by shutting down immunocompetent cells through CD46-generated T-cells. Herein, we review this pathway for the induction of Tregs, focusing on a role for the complement system and especially signaling through CD46 on human T-cells.     

Increased sensitivity of early apoptotic cells to complement-mediated lysis

Opsonization of apoptotic cells with complement proteins contributes to their clearance by phagocytes. Little is known about the lytic effects of complement on apoptotic cells. Sensitivity of cells treated with anti-Fas antibody (Jurkat cells), staurosporine or etoposide (Raji cells) to lysis by complement was examined. As shown here, early apoptotic cells are more sensitive to lysis by antibody and complement than control cells. More complement C3 and C9 bound to apoptotic than to control cells, even though antibody binding was similar. Enhanced killing and C3/C9 deposition were blocked by benzyloxy-Val-Ala-Asp-fluoromethylketone, a pan-caspase inhibitor. Complement-mediated lysis of early apoptotic cells was also prevented by inhibitors of caspases 6, 8, 9 or 10. In contrast, caspase inhibitors had no effect on the lysis of non-apoptotic Jurkat and Raji cells. Early apoptotic Jurkat cells were also more sensitive to lysis by the pore formers streptolysin O and melittin. Sensitivity of Jurkat Bcl-2 transfectants to lysis by complement was analyzed. Enhanced Bcl-2 expression was associated with reduced C3 deposition and lower sensitivity to complement-mediated lysis. These results demonstrate that at an early stage in apoptosis, following caspase activation, cells become sensitive to necrotic-type death by complement and other pore formers. Furthermore, they suggest that Bcl-2 is actively protecting Jurkat cells from complement-mediated lysis.     

Enhanced sensitivity of P-glycoprotein-positive multidrug resistant tumor cells to complement-mediated lysis

The interaction of KB-V1, a multidrug resistant (MDR) variant of the KB-3–1 human oral carcinoma, with human complement was investigated. KB-V1 cells were found to be more sensitive than KB-3–1 cells to complement-mediated lysis. Detailed analysis of the capacity of KB cells to activate human complement demonstrated that both C3b deposition and formation of the membrane attack complex (MAC) are higher on KB-V1 than on KB-3–1 cells. Furthermore, the MAC formed on KB-V1 cells, but not on KB-3–1 cells, was found to be resistant to trypsin treatment, i.e. more stably inserted into the plasma membrane. Immunofluorescence analysis by flow cytometry showed that KB-V1 cells express less decay-accelerating factor (DAF, CD55) than KB-3–1 cells. Two other complement regulatory proteins, membrane cofactor protein (MCP, CD46) and CD59 are expressed to a similar extent on both KB-V1 and KB-3–1 cells. Treatment of KB-V1 cells with neutralizing anti-P-glycoprotein (P-gp) monoclonal antibodies reduced their sensitivity to complement. In addition, KB-V1 revertants which cease to express P-gp become more resistant to complement. These results indicate that multiple factors, such as reduced expression of DAF, enhanced deposition of C3b and increased binding and stability of the MAC may contribute to the increased complement sensitivity of KB-V1 cells. It is suggested that P-gp is responsible for the complement-sensitive phenotype of KB-V1 cells.     

Antigen-presenting cell exosomes are protected from complement-mediated lysis by expression of CD55 and CD59

Exosomes are secreted nanometer-sized vesicles derived from antigen-presenting cells, which have attracted recent interest as they likely play important roles in immune regulation, and their use as cell-free tools for immunotherapy has been proposed. Liposomes used clinically as transport vehicles can activate the complement system, leading to their rapid degradation and significant inflammatory toxicity. The use of isolated exosomes in therapy, therefore, may also elicit complement activation, reducing their potential efficacy. We have examined the expression and functional roles of the membrane regulators of complement (CD46, CD55 and CD59) on antigen-presenting cell-derived exosomes. Exosomes express the glycosylphosphatidylinositol (GPI)-anchored regulators CD55 and CD59, but not the transmembrane protein CD46. Antibody blocking of CD55 in the presence of sensitizing antibody (w6/32) and human serum resulted in increased C3b deposition and significantly increased exosome lysis. Blockade of CD59 also resulted in significant lysis, while blocking both CD55 and CD59 increased lysis still further. We conclude that exosomes express GPI-anchored complement regulators in order to permit their survival in the extracellular environment.     

Measles virus attachment proteins with impaired ability to bind CD46 interact more efficiently with the homologous fusion protein

Fusion promotion by measles virus (MV) depends on an interaction between the hemagglutinin (H) and fusion (F) glycoproteins. Amino acid substitutions in MV H that drastically reduce hemagglutinating activity result in an increase in the amount of H (primarily the 74 kDa isoform) detectable in a complex with F at the cell surface. This is in direct contrast to the loss of the ability to detect a complex between the fusion protein of Newcastle disease virus and most attachment proteins that lack receptor binding activity. These opposing results provide support for the existence of different mechanisms for the regulation of fusion by these two paramyxoviruses.     

A functional analysis of recombinant soluble CD46 in vivo and a comparison with recombinant soluble forms of CD55 and CD35 in vitro

The human cell surface complement regulatory proteins CD46 (MCP), CD55 (DAF) and CD35 (CR1) protect autologous cells from complement-mediated damage by inhibiting C3 and C5 convertases. This regulatory potential has previously been exploited in the treatment of some models of inflammatory injury by the generation of recombinant soluble (rs) proteins, such as rsCD55 and rsCD35. More recently, we have shown that rsCD46 inhibits complement activation in the fluid phase. In this report, the ability of rsCD46, rsCD55 and rsCD35 to regulate human complement activation mediated by the classical and alternative pathways was directly compared. Regulation of the classical pathway in vitro was clearly demonstrated by all three soluble proteins; however, rsCD35 was a more effective inhibitor than either rsCD46 or rsCD55. A combination of rsCD46 + rsCD55 was more potent than either of these proteins alone. Cell lysis via alternative pathway activation in vitro was efficiently regulated by rsCD46 and rsCD35 to a similar extent, whereas rsCD55 was not as effective. Assays of rsCD46 in vivo have previously not been possible due to difficulties in expressing sufficient quantities of protein. This limitation has been overcome and now we report the ability of rsCD46 to inhibit immune complex-mediated inflammation in a rat using the reverse passive Arthus reaction model. Administration of rsCD46 significantly reduced the size of lesion, and histological examination showed a reduction in inflammatory infiltrate and edema. These data suggest that rsCD46, in addition to rsCD55 and rsCD35, may be a useful therapeutic agent.     

Enhanced MHC class II-restricted presentation of measles virus (MV) hemagglutinin in transgenic mice expressing human MV receptor CD46

This study analyzes the role of the measles virus (MV) receptor, i.e. the human CD46 molecule, in the MHC class II-restricted presentation of MV hemagglutinin (H). We generated transgenic mice ubiquitously expressing CD46, with a similar level of transgene expression on the surface of antigen-presenting cells (APC), i.e. B cells, dendritic cells (DC) and macrophages. APC isolated from transgenic mice and nontransgenic controls were tested for their ability to present MV H to H-specific CD4⁺ I-E ^d -restricted T cell hybridomas. All three populations of APC were capable of presenting MV to T cell hybridomas, DC being the most efficient. Expression of CD46 on B lymphocytes increased MHC class II-dependent presentation of MV H up to 100-fold, while CD46-transgenic DC stimulated H-specific T cell hybridomas up to 10-fold better than nontransgenic DC. Interestingly, expression of CD46 did not change the presentation efficiency of transgenic macrophages, indicating that CD46-dependent enhancement of antigen presentation depends on the nature of the APC. Furthermore, a single injection of UV-inactivated MV particles into CD46-transgenic mice, but not nontransgenic controls, induced generation of MV-specific T lymphocytes and production of anti-H antibodies, suggesting a role for CD46 in the efficient capture of MV in vivo. These results show for the first time that one ubiquitously expressed cell surface receptor, like CD46, could function in receptor-mediated antigen presentation both in vitro and in vivo and its performance depends on the type of APC which expresses it.     

Measles virus interacts with human SLAM receptor on dendritic cells to cause immunosuppression

Measles virus (MV) infects dendritic cells (DCs) resulting in immunosuppression. Human DCs express two MV receptors: CD46 and human signaling lymphocyte activation molecule (hSLAM); thus, the role played by either alone is unclear. Because wild-type (wt) MV uses hSLAM receptor preferentially, we dissected the molecular basis of MV-DC interaction and resultant immunosuppression through the hSLAM receptor by creating transgenic (tg) mice expressing hSLAM on DCs. After infection with wt MV, murine splenic DCs expressing hSLAM receptor had less B7-1, B7-2, CD40, MHC class I, and MHC class II molecules on their surfaces and displayed an increased rate of apoptosis when compared to uninfected DCs. Further, MV-infected DCs failed to stimulate allogeneic T cells and inhibited mitogen-dependent T-cell proliferation. Individual expression of human SLAM, interferon alpha/beta receptor, tumor necrosis factor-alpha, and lymphotoxin-alpha or beta from T cells was not required for MV-infected DCs to inhibit the proliferation of T cells.     

The differentiation of human memory B cells into specific antibody-secreting cells is CD40 independent

It is generally accepted that memory B cells can be defined by their ability to produce, upon antigenic challenge, somatically mutated antibody molecules characterized by an increased affinity and by the expression of a downstream heavy chain isotype. However, the inability to isolate this particular B cell compartment has precluded the study of memory B lymphocyte physiology in man. We previously reported on the identification of an IgD^? B cell subset in human tonsils that we defined as CD38^? B cells, whose phenotype is highly reminiscent of that of memory B lymphocytes from the splenic marginal zone of rodents. In the present study, we developed a model of the measles virus (MV)-specific secondary antibody response in vitro to assess the presence of memory B lymphocytes in different B cell subsets isolated from human tonsils and explore the activation requirements of human memory B cells. Our findings show that the memory B cell pool resides in the CD38^? B cell subpopulation and that the differentiation of MV-activated memory B cells into antibody-secreting cells can be achieved upon co-stimulation with interleukin (IL)-2 and IL-10, but does not require engagement of CD40. Interestingly, the CD40-mediated signal was found to synergize with Ig-cross-linking agents for the proliferation of memory B cells, but strongly suppressed their capacity to differentiate along the plasmacytoid pathway. Collectively, our results suggest that the CD40 signaling pathway is instrumental for the clonal expansion of the memory B cell pool, but does not operate in the later phase of the response, which allows their maturation into antibody-secreting cells.     

Transfer of prostasomal CD59 to CD59-deficient red blood cells results in protection against complement-mediated hemolysis

PROBLEM: Prostasomes isolated from human seminal plasma have complement regulatory properties because of their content of CD59, a glycosylphosphatidylinositol (GPI)-anchored protein. We investigated a functional role of prostasomes by the possibility of transferring CD59 from prostasomes to rabbit erythrocytes (RE) and human erythrocytes obtained from patients with paroxysmal nocturnal hemoglobinuria (PNH), both types of cells lacking CD59. METHOD OF STUDY: We used the assay of hemolytic activity of the alternative pathway of the complement system to compare the liability of the erythrocytes to hemolysis by the complement system with and without pre-incubation with prostasomes. CD59 gained by the RE and PNH erythrocytes was established by flow cytometry. The effect of phosphatidylinositol phospholipase C (PIPLC) on the GPI anchor of prostasomal CD59 and the effect of heat treatment on the prostasomes were also studied. Anti-CD59 antibodies were used to block the protective effect of prostasomes on erythrocytes. RESULTS: Both RE and PNH erythrocytes showed diminished complement-mediated hemolysis after incubation with prostasomes. This was because of the transfer of CD59 from prostasomes to the red blood cells during pre-incubation as evidenced by the hemolytic assay and flow-cytometry. The efficacy of the prostasomes was affected by heat treatment and was totally lost at 100 degrees C. Phosphatidylinositol phospholipase C broke the GPI anchor and released CD59 from prostasomes and the RE surface (after pre-incubation with prostasomes) but not from the human PNH erythrocytes. CONCLUSIONS: A transfer mechanism of CD59 takes place during pre-incubation from prostasomes to erythrocytes lacking CD59 which supports the idea that transfer of prostasomal CD59 can protect cells from lysis elicited by C5b-9. This might be a mechanism by which autologous and allogeneic cells are protected against complement attack in the genital tracts.     

Adenovirus interactions with CD46 on transgenic mouse erythrocytes

Hemagglutination is an established method but has not been used previously to determine the efficacy of virus binding to a specific cellular receptor. Here we have utilized CD46-expressing erythrocytes from a transgenic mouse to establish whether and to what extent the species B adenoviruses (Ads) as well as Ad37 and Ad49 of species D can interact with CD46. A number of different agglutination patterns, and hence CD46 interactions, could be observed for the different adenovirus types. In this system Ad7p, Ad11a, and Ad14 did not agglutinate mouse erythrocytes at all. Hemagglutination of CD46 expressing erythrocytes with high efficiency was observed for the previously established CD46 users Ad11p and Ad35 as well as for the less investigated Ad34. Ad50 agglutinated with moderate efficiency. Ad16, Ad21 and Ad49 gave incomplete agglutination. Ad16 was the only adenovirus that could be eluted. No specific CD46 interaction could be observed for Ad3p or for Ad37.     

In vivo depletion of CD4CD25 regulatory T cells is associated with improved antiviral responses in cats chronically infected with feline immunodeficiency virus

Regulatory T (Treg) cells are activated and suppress immune responses during infection, and are characterized as CD4⁺CD25^hiFOXP3⁺. Ex vivo studies demonstrate that Treg cells potentially suppress anti-HIV-1 T cell responses. Lentivirus-induced CD4⁺CD25^hi Treg cells were first described in feline immunodeficiency virus (FIV)-infected cats. In the present study we demonstrate that anti-feline CD25 monoclonal antibody (mAb) therapy depletes Treg cells in FIV-infected cats for 4 weeks and does not exacerbate viral replication or proinflammatory cytokine production. Significant FIV-specific immune responses are revealed in Treg cell-depleted cats. These anti-FIV effector cells exist prior to Treg cell depletion and are not expanded while Treg cells are depleted. Importantly, cats receiving the Treg cell-depleting mAb are able to produce a robust humoral response to new antigen. We propose that short-term in vivo Treg cell depletion during chronic HIV-1 infection could provide a window of opportunity for therapeutic vaccination in individuals with controlled viral replication.     

Defining the role of CD46, CD80 and CD86 in mediating adenovirus type 3 fiber interactions with host cells

Attachment of human adenoviruses (Ads) to host cells is mediated by the interaction of the fiber protein of the capsid with specific cell-surface molecules. For one of the species B adenoviruses, Ad3, the mechanism of binding to cells remains to be defined. Several previous reports have proposed CD46, CD80 or CD86 as possible Ad3 fiber attachment molecules. In this study, CD80 and CD86 were not found to mediate Ad3 fiber binding or Ad3-EGFP transduction of cells. Low levels of Ad3-EGFP transduction of a CHO cell line expressing relatively high levels of CD46 were detected which might suggest a role for CD46 in facilitating Ad3: cell interactions, in the absence of other attachment molecules. Anti-CD46 antibodies and siRNAs had almost no effect on Ad3 fiber binding or Ad3-EGFP transduction of HeLa cells. However, treatment of A549 cells with CD46 siRNA resulted in some decrease of transduction with Ad3-EGFP.     

可溶性嵌合补体抑制分子的构建及表达

目的：制备能抑制补体活化的2个关键环节（C3/C5转化酶和MAC的形成）的一种新型、高效补体抑制剂。方法：先设计引物，然后通过PCR技术扩增重组可溶性CD46/CD55/CD59嵌合分子cDNA片段，重组于pSecTagA真核表达载体上，分别利用COS-7细胞和中国仓鼠卵巢（Chinese hamster ovary，CHO）细胞进行表达，并用抗人CD46多克隆抗体及抗人CD55、CD59单克隆抗体对表达产物进行western bolting鉴定。结果：DNA测序证实，前端带有小鼠IgGκ链前导序列，后端融合有编码6个组氨酸碱基的CD46/CD55/CD59嵌合分子cDNA片段的阅读框完整。免疫印迹结果显示，表达的重组蛋白分别能与抗人CD46多克隆抗体和抗人CD55、CD59单克隆抗体结合。结论：成功构建并在真核细胞内表达了重组可溶性CD46/CD55/CD59嵌合分子，为进一步研制和开发新一代多功能、多靶点补体抑制剂奠定了基础。     

CD4 T cell control primary measles virus infection of the CNS: regulation is dependent on combined activity with either CD8 T cells or with B cells: CD4, CD8 or B cells alone are ineffective

Measles virus (MV), one of the most infectious of human pathogens, still infects over 30 million humans and causes over 500,000 deaths each year [Griffin, D., 2001. Measles virus. In: Fields, B., Knipe, D., Howley, P. (Eds.), Fields Virology. Lippincott-Raven, Philadelphia, pp. 1401-1442; ]. Death is primarily due to secondary microbial infections associated with the immunosuppression caused by MV. Studies of humans with genetic or acquired deficiencies of either the humoral or cellular arm of the immune system, and rodent models have implicated T cells in the control of the ongoing MV infection but the precise role and activities of the specific T cell subset or the molecules they produce is not clear. Using a transgenic mouse model in conjunction with depletion and reconstitution of individual B and T cell subsets alone or in combination, we show that neither CD4, CD8 nor B cells per se control acute MV infection. However, combinations of either CD4 T cells and B cells, or of CD4 and CD8 T cells are essential but CD8 T with B cells are ineffective. Interferon-gamma and neutralizing antibodies, but neither perforin nor TNF-alpha alone are associated with clearance of MV infection. TNF-alpha combined with interferon-gamma is more effective in protection than interferon alone. Further, the lack of an interferon-gamma response leads to persistence of MV.     

Protection of human breast cancer cells from complement-mediated lysis by expression of heterologous CD59

CD59, decay accelerating factor (DAF) and membrane cofactor protein (MCP) are widely expressed cell surface glycoproteins that protect host cells from the effects of homologous complement attack. Complement inhibitory activity of these proteins is species-selective. We show that the human breast cancer cell line MCF7 is relatively resistant to lysis by human complement, but is effectively lysed by rat or mouse complement. CD59, DAF and MCP were all shown to be expressed by MCF7. The species-selective nature of CD59 activity was used to demonstrate directly the effectiveness of CD59 at protecting cancer cells from complement-mediated lysis. cDNAs encoding rat and mouse CD59 were separately transfected into MCF7 cells, and cell populations expressing high levels of the rodent CD59 were isolated by cell sorting. Data show that rat and mouse CD59 were highly effective at protecting transfected MCF7 cells from lysis by rat and mouse complement, respectively. Data further reveal that rat CD59 is not effective against mouse complement, whereas mouse CD59 is effective against both mouse and rat complement. These studies establish a model system for relevant in vivo studies aimed at determining the effect of complement regulation on tumourigenesis, and show that for effective immunotherapy using complement-activating anti-tumour antibodies, the neutralization of CD59 and/or other complement inhibitory molecules will probably be required.     

Dysregulated CD46 shedding interferes with Th1‐contraction in systemic lupus erythematosus

IFN‐γ‐producing T helper 1 (Th1) cell responses mediate protection against infections but uncontrolled Th1 activity also contributes to a broad range of autoimmune diseases. Autocrine complement activation has recently emerged as key in the induction and contraction of human Th1 immunity: activation of the complement regulator CD46 and the C3aR expressed by CD4⁺ T cells via autocrine generated ligands C3b and C3a, respectively, are critical to IFN‐γ production. Further, CD46‐mediated signals also induce co‐expression of immunosuppressive IL‐10 in Th1 cells and transition into a (self)‐regulating and contracting phase. In consequence, C3 or CD46‐deficient patients suffer from recurrent infections while dysregulation of CD46 signaling contributes to Th1 hyperactivity in rheumatoid arthritis and multiple sclerosis. Here, we report a defect in CD46‐regulated Th1 contraction in patients with systemic lupus erythematosus (SLE). We observed that MMP‐9‐mediated increased shedding of soluble CD46 by Th1 cells was associated with this defect and that inhibition of MMP‐9 activity normalized release of soluble CD46 and restored Th1 contraction in patients’ T cells. These data may deliver the first mechanistic explanation for the increased serum CD46 levels observed in SLE patients and indicate that targeting CD46‐cleaving proteases could be a novel avenue to modulate Th1 responses.     

CD46‐induced human Treg enhance B‐cell responses

Regulatory CD4⁺ T cells (Treg) are important modulators of the immune response. Different types of Treg have been identified based on whether they are thymically derived (natural Treg) or induced in the periphery (adaptive Treg). We recently reported on an adaptive Treg phenotype that can be induced by the concomitant stimulation of human CD4⁺ T cells through CD3 and the membrane complement regulator CD46. These complement (CD46)‐induced regulatory T cells (cTreg) potently inhibit bystander T‐cell proliferation through high‐level secretion of IL‐10. In addition, cTreg express granzyme B and exhibit cytotoxic effects toward activated effector T cells. Here, we analyzed the effect of cTreg on B‐cell functions in a co‐culture system. We found that cTreg enhance B‐cell Ab production. This B‐cell support is dependent on cell/cell contact as well as cTreg‐derived IL‐10. In addition, we show that T cells from a CD46‐deficient patient are not capable of promoting B‐cell responses, whereas CD46‐deficient B cells have no intrinsic defect in Ig production. This finding may relate to a subset of CD46‐deficient patients, who present with common variable immunodeficiency. Thus, the lack of cTreg function in optimizing B‐cell responses could explain why some CD46‐deficient patients develop common variable immunodeficiency.     

Functional characterization of two novel non-synonymous alterations in CD46 and a Q950H change in factor H found in atypical hemolytic uremic syndrome patients

Atypical hemolytic uremic syndrome (aHUS) is a disease of complement dysregulation, characterized by hemolytic anemia, thrombocytopenia and acute renal failure. Mutations in complement inhibitors are major risk factors for development of aHUS. The three aHUS patients reported in this study had several previously identified alterations in complement inhibitors; e.g. risk haplotypes in CD46 and factor H but we also identified two novel heterozygous non-synonymous CD46 alterations (p.E142Q and p.G259V). Presence of G259V caused decreased expression of the recombinant mutant CD46 compared to wild type (WT). Western blot analysis showed that the majority of the expressed G259V protein was in the precursor form, suggesting that it is processed less efficiently than WT. Low CD46 expression on the surface of the patient's neutrophils confirmed the in vitro results. Further, G259V had a substantially impaired ability to act as a cofactor to factor I, in the degradation of both C3b and C4b. The E142Q mutant showed neither decreased expression nor impaired function. Two of the patients also had a heterozygous non-synonymous alteration in factor H (p.Q950H), reported previously in aHUS but not functionally tested. This variant showed moderately impaired function in hemolytic assays, both using patient sera and recombinant proteins. The recombinant Q950H also showed a somewhat decreased expression compared to WT but the complement inhibitory function in fluid phase was normal. Taken together, we report a novel CD46 alteration showing both a decreased protein expression and substantially impaired cofactor function (G259V) and another without an effect on expression or cofactor function (E142Q). Moreover, mild consequences of a previously reported aHUS associated rare variant in factor H (Q950H) was also revealed, underlining the clear need for functional characterization of each new aHUS associated mutation.