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The idea of designing multispecific antibodies capable of simultaneously engaging two or more epitopes on the same or different antigens was developed more than 50 years ago. However, the molecular complexity of such molecules may pose significant challenges for their development and clinical use. Particularly challenging is to obtain the correctly assembled combination of different polypeptide chains, which places significant demand on downstream process development, analytical characterization and control strategy. Here, we review the progress made in protein engineering to force the correct assembly of different heavy and light chains, as well as upstream and downstream processes currently applied to control generation of unwanted byproduct species. We cover in-depth the analytical methods available to characterize such complex molecules, focusing on mispairing analysis and functional characterization.

AICL glycoproteins are cognate activation-induced ligands of the C-type lectin-like receptor NKp80, which is expressed on virtually all mature human NK cells, and NKp80–AICL interaction stimulates NK cell effector functions such as cytotoxicity and cytokine secretion. Notably, AICL and NKp80 are encoded by adjacent genes in the NK gene complex and are coexpressed by human NK cells. Whereas AICL is intracellularly retained in resting NK cells, exposure of NK cells to proinflammatory cytokines results in AICL surfacing and susceptibility to NKp80-mediated NK fratricide. In this study, we characterize molecular determinants of AICL glycoproteins that cause intracellular retention, thereby controlling AICL surface expression. Cys87 residing within the C-type lectin-like domain not only ensures stable homodimerization of AICL glycoproteins by disulfide bonding, but Cys87 is also required for efficient cell surface expression of AICL homodimers and essential for AICL–NKp80 interaction. In contrast, cytoplasmic lysines act as negative regulators targeting AICL for proteasomal degradation. One atypical and three conventional N-linked glycosylation sites in the AICL C-type lectin-like domain critically impact maturation and surfacing of AICL, which is strictly dependent on glycosylation of at least one conventional glycosylation site. However, although the extent of conventional N-linked glycosylation positively correlates with AICL surface expression, the atypical glycosylation site impairs AICL surfacing. Stringent control of AICL surface expression by glycosylation is reflected by the pronounced interaction of AICL with calnexin and the impaired AICL expression in calnexin-deficient cells. Collectively, our data demonstrate that AICL expression and surfacing are tightly controlled by several independent cellular posttranslational mechanisms.

The mouse gut epithelium represents a constitutively challenged environment keeping intestinal commensal microbiota at bay and defending against invading enteric pathogens. The complex immunoregulatory network of the epithelial barrier surveillance also involves NK gene complex (NKC)–encoded C-type lectin-like molecules such as NKG2D and Nkrp1 receptors. To our knowledge, in this study, we report the first characterization of the orphan C-type lectin-like molecule Clr-a encoded by the Clec2e gene in the mouse NKC. Screening of a panel of mouse tissues revealed that Clec2e transcripts are restricted to the gastrointestinal tract. Using Clr-a–specific mAb, we characterize Clr-a as a disulfide-linked homodimeric cell surface glycoprotein. Of note, a substantial fraction of Clr-a molecules are retained intracellularly, and analyses of Clr-a/Clr-f hybrids attribute intracellular retention to both the stalk region and parts of the cytoplasmic domain. Combining quantitative PCR analyses with immunofluorescence studies revealed exclusive expression of Clr-a by intestinal epithelial cells and crypt cells throughout the gut. Challenge with polyinosinic-polycytidylic acid results in a rapid and strong downregulation of intestinal Clr-a expression in contrast to the upregulation of Clr-f, a close relative of Clr-a, that also is specifically expressed by the intestinal epithelium and acts as a ligand of the inhibitory Nkrp1g receptor. Collectively, we characterize expression of the mouse NKC-encoded glycoprotein Clr-a as strictly associated with mouse intestinal epithelium. Downregulation upon polyinosinic-polycytidylic acid challenge and expression by crypt cells clearly distinguish Clr-a from the likewise intestinal epithelium-restricted Clr-f, pointing to a nonredundant function of these highly related C-type lectin-like molecules in the context of intestinal immunosurveillance.

The natural cytotoxicity receptor (NCR) NKp30 (CD337) is a key player for NK cell immunosurveillance of infections and cancer. The molecular details of ligand recognition and its connection to CD3ζ signaling remain unsolved. Here, we show that the stalk domain (129KEHPQLGAGTVLLLR143) of NKp30 is very sensitive to sequence alterations, as mutations lead to impaired ligand binding and/or signaling capacity. Surprisingly, the stalk domains of NKp30 and NKp46, another NCR employing CD3ζ for signaling, were not exchangeable without drastic deficiencies in folding, plasma membrane targeting, and/or ligand-induced receptor signaling. Further mutational studies, N-glycosylation mapping, and plasma membrane targeting studies in the absence and presence of CD3ζ suggest two interconvertible types of NCR-CD3ζ assemblies: 1) a signaling incompetent structural NKp30-CD3ζ complex and 2) a ligand-induced signaling competent NKp30-CD3ζ complex. Moreover, we propose that ligand binding triggers translocation of Arg-143 from the membrane interface into the membrane to enable alignment with oppositely charged aspartate residues within CD3ζ and activation of CD3ζ-signaling.

International audience; NKp46 (CD335) is a surface receptor shared by both human and mouse natural killer (NK) cells and innate lymphoid cells (ILCs) that transduces activating signals necessary to eliminate virus-infected cells and tumors. Here, we describe a spontaneous point mutation of cysteine to arginine (C14R) in the signal peptide of the NKp46 protein in congenic Ly5.1 mice and the newly generated NCR(B6C14R) strain. Ly5.1(C14R) NK cells expressed similar levels of Ncr1 mRNA as C57BL/6, but showed impaired surface NKp46 and reduced ability to control melanoma tumors in vivo. Expression of the mutant NKp46(C14R) in 293T cells showed that NKp46 protein trafficking to the cell surface was compromised. Although Ly5.1(C14R) mice had normal number of NK cells, they showed an increased number of early maturation stage NK cells. CD49a(+)ILC1s were also increased but these cells lacked the expression of TRAIL. ILC3s that expressed NKp46 were not detectable and were not apparent when examined by T-bet expression. Thus, the C14R mutation reveals that NKp46 is important for NK cell and ILC differentiation, maturation and function. Significance Innate lymphoid cells (ILCs) play important roles in immune protection. Various subsets of ILCs express the activating receptor NKp46 which is capable of recognizing pathogen derived and tumor ligands and is necessary for immune protection. Here, we describe a spontaneous point mutation in the signal peptide of the NKp46 protein in congenic Ly5.1 mice which are widely used for tracking cells in vivo. This Ncr1 C14R mutation impairs NKp46 surface expression resulting in destabilization of Ncr1 and accumulation of NKp46 in the endoplasmic reticulum. Loss of stable NKp46 expression impaired the maturation of NKp46(+) ILCs and altered the expression of TRAIL and T-bet in ILC1 and ILC3, respectively.

The activating natural cytotoxicity receptors on natural killer (NK) cells play a fundamental role in immunosurveillance of infections and cancer. Phylogenetic analyses showed that NKp30 is highly conserved in almost all jawed vertebrates and thus, represents one of the most ancient NK cell receptors. However, in contrast to other higher vertebrates, NKp30 is only a pseudogene in mouse, which contains two premature stop codons. To decipher the evolutionary role and biological function of NKp30 in mouse, we removed these premature stop codons and expressed the putative mouse NKp30 (mNKp30) protein as soluble Fc fusion construct and as full-length receptor on A5-GFP reporter cells. Interestingly, even though both NKp30 variants were expressed, maturation and targeting to the plasma membrane were impaired. Previous studies implicated that N-linked glycosylation is crucial for plasma membrane targeting and ligand binding of human NKp30. However, even though present in all other jawed vertebrates analyzed so far, these three N-linked glycosylation sites are missing in mouse NKp30. Interestingly, reconstitution of N-linked glycosylation enabled secretion of a mNKp30-Fc fusion protein which recognized a yet unknown ligand on the plasma membrane of mastocytoma cells. Based on these data, our study is the first to show expression and functional analysis of a mNKp30 protein suggesting that the mouse NKp30 pseudogene is the result of a species-specific loss of function.

Head and neck squamous cell carcinoma (HNSCC) is a highly heterogeneous and aggressive tumor originating from the epithelial lining of the upper aero-digestive tract accounting for 300,000 annual deaths worldwide due to failure of current therapies. The natural killer group 2D (NKG2D) receptors on natural killer (NK) cells and several T cell subsets play an important role for immunosurveillance of HNSCC and are thus targeted by tumor immune evasion strategies in particular by shedding of various NKG2D ligands (NKG2DLs). Based on plasma and tumor samples of 44 HNSCC patients, we found that despite compositional heterogeneity the total plasma level of NKG2DLs correlates with NK cell inhibition and disease progression. Strikingly, based on tumor spheroids and primary tumors of HNSCC patients, we found that NK cells failed to infiltrate HNSCC tumors in the presence of high levels of NKG2DLs, demonstrating a novel mechanism of NKG2DL-dependent tumor immune escape. Therefore, the diagnostic acquisition of the plasma level of all NKG2DLs might be instrumental for prognosis and to decipher a patient cohort, which could benefit from restoration of NKG2D-dependent tumor immunosurveillance. Along these lines, we could show that removal of shed NKG2DLs (sNKG2DLs) from HNSCC patients’ plasma restored NK cell function in vitro and in individual patients following surgical removal of the primary tumor. In order to translate these findings into a therapeutic setting, we performed a proof-of-concept study to test the efficacy of adsorption apheresis of sNKG2DLs from plasma after infusion of human MICA in rhesus monkeys. Complete removal of MICA was achieved after three plasma volume exchanges. Therefore, we propose adsorption apheresis of sNKG2DLs as a future preconditioning strategy to improve the efficacy of autologous and adoptively transferred immune cells in cellular cancer immunotherapy.

Killer immunoglobulin-like receptors (KIRs) represent a highly polymorphic and diverse gene family in rhesus macaques. Analyses of the respective gene products have been hampered until now due to non-availability of specific monoclonal antibodies and failure of cross-reactivity of anti-human KIR antibodies. We utilised one activating (KIR3DSW08) and two inhibitory (KIR3DLW03 and KIR3DL05) rhesus macaque KIR-Fc fusion proteins for generation of monoclonal antibodies in mice. Besides broadly reacting ones, we obtained anti-rhesus macaque KIR antibodies with intermediate and with single specificity. These monoclonal antibodies were tested for binding to a panel of rhesus macaque KIR proteins after heterologous expression on transiently transfected cells. Epitope mapping identified two polymorphic regions that are located next to each other in the mature KIR proteins. The availability of monoclonal antibodies against rhesus macaque KIR proteins will enable future studies on KIR at the protein level in rhesus macaques as important animal models of human infectious diseases. Electronic supplementary material The online version of this article (doi:10.1007/s00251-012-0640-2) contains supplementary material, which is available to authorized users.

Immunosuppressive factors, such as soluble major histocompatibility complex class I chain-related peptide A (sMICA) and transforming growth factor beta 1 (TGF-beta1), are involved in tumour immune escape mechanisms (TIEMs) exhibited by head and neck squamous cell carcinomas (HNSCCs) and may represent opportunities for therapeutic intervention. In order to overcome TIEMs, we investigated the antibody-dependent cellular cytotoxicity (ADCC), cytokine release and retargeted tumour-infiltration of sMICA-inhibited patient NK cells expressing Fcγ receptor IIIa (FcγRIIIa, CD16a) in the presence of cetuximab, an anti-EGFR (epidermal growth factor receptor, HER1) monoclonal antibody (mAb). Compared to healthy controls, relapsed HNSCC patients (n = 5) not currently in treatment revealed decreased levels of circulating regulatory NK cell subsets in relation to increased cytotoxic NK cell subpopulations. Elevated sMICA and TGF-beta1 plasma levels correlated with diminished TNFalpha and IFN-gamma release and decreased NKG2D (natural killer group 2 member D)-dependent killing of HNSCC cells by NK cells. Incubation of IL-2-activated patient NK cells with patient plasma containing elevated sMICA or sMICA analogues (shed MICA [shMICA], recombinant MICA [rMICA]) significantly impaired NKG2D-mediated killing by down-regulation of NKG2D surface expression. Of note, CD16 surface expression levels, pro-apoptotic and activation markers, and viability of patient and healthy donor NK cell subpopulations were not affected by this treatment. Accordingly, cetuximab restored killing activity of sMICA-inhibited patient NK cells against cetuximab-coated primary HNSCC cells via ADCC in a dose-dependent manner. Rapid reconstitution of anti-tumour recognition and enhanced tumour-infiltration of treated NK cells was monitored by 24 h co-incubation of HNSCC tumour spheroids with cetuximab (1 µg/ml) and was characterised by increased IFN-gamma and TNFalpha secretion. This data show that the impaired NK cell-dependent tumour-surveillance in relapsed HNSCC patients could be reversed by the re-establishment of ADCC-mediated effector cell activity, thus supporting NK cell-based immunotherapy in combination with antineoplastic monoclonal mAbs.

In the case of a release of highly pathogenic bacteria (HPB), there is an urgent need for rapid, accurate, and reliable diagnostics. MALDI-TOF mass spectrometry is a rapid, accurate, and relatively inexpensive technique that is becoming increasingly important in microbiological diagnostics to complement classical microbiology, PCR, and genotyping of HPB. In the present study, the results of a joint exercise with 11 partner institutions from nine European countries are presented. In this exercise, 10 distinct microbial samples, among them five HPB, Bacillus anthracis , Brucella canis , Burkholderia mallei , Burkholderia pseudomallei , and Yersinia pestis , were characterized under blinded conditions. Microbial strains were inactivated by high-dose gamma irradiation before shipment. Preparatory investigations ensured that this type of inactivation induced only subtle spectral changes with negligible influence on the quality of the diagnosis. Furthermore, pilot tests on nonpathogenic strains were systematically conducted to ensure the suitability of sample preparation and to optimize and standardize the workflow for microbial identification. The analysis of the microbial mass spectra was carried out by the individual laboratories on the basis of spectral libraries available on site. All mass spectra were also tested against an in-house HPB library at the Robert Koch Institute (RKI). The averaged identification accuracy was 77% in the first case and improved to >93% when the spectral diagnoses were obtained on the basis of the RKI library. The compilation of complete and comprehensive databases with spectra from a broad strain collection is therefore considered of paramount importance for accurate microbial identification.

Background The complex cellular networks within tumors, the cytokine milieu, and tumor immune escape mechanisms affecting infiltration and anti-tumor activity of immune cells are of great interest to understand tumor formation and to decipher novel access points for cancer therapy. However, cellular in vitro assays, which rely on monolayer cultures of mammalian cell lines, neglect the three-dimensional architecture of a tumor, thus limiting their validity for the in vivo situation. Methods Three-dimensional in vivo-like tumor spheroid were established from human cervical carcinoma cell lines as proof of concept to investigate infiltration and cytotoxicity of NK cells in a 96-well plate format, which is applicable for high-throughput screening. Tumor spheroids were monitored for NK cell infiltration and cytotoxicity by flow cytometry. Infiltrated NK cells, could be recovered by magnetic cell separation. Results The tumor spheroids were stable over several days with minor alterations in phenotypic appearance. The tumor spheroids expressed high levels of cellular ligands for the natural killer (NK) group 2D receptor (NKG2D), mediating spheroid destruction by primary human NK cells. Interestingly, destruction of a three-dimensional tumor spheroid took much longer when compared to the parental monolayer cultures. Moreover, destruction of tumor spheroids was accompanied by infiltration of a fraction of NK cells, which could be recovered at high purity. Conclusion Tumor spheroids represent a versatile in vivo-like model system to study cytotoxicity and infiltration of immune cells in high-throughput screening. This system might proof useful for the investigation of the modulatory potential of soluble factors and cells of the tumor microenvironment on immune cell activity as well as profiling of patient-/donor-derived immune cells to personalize cellular immunotherapy. Electronic supplementary material The online version of this article (doi:10.1186/s12885-015-1321-y) contains supplementary material, which is available to authorized users.

Competitive or cooperative behaviors of children were observed in two experiments. In Experiment 1, 7- to 9-year-old boys and girls played a board game with same- or opposite-sex partners under instructions which stressed either group or individual performance. Girls' behavior varied as a function of both instructions and partner sex. Girls playing the game with boys showed more highly competitive behavior than did girls who played the game with girls. Boys were highly competitive under all conditions. In Experiment 2, preschool children were observed in a similar game. For both sexes, children who played the game with opposite-sex partners were more competitive and obtained fewer rewards than those who interacted with same-sex partners. Thus, with development, boys show a change from selective competitiveness to a very general tendency to compete, while girls show a consistent tendency to vary their game behavior according to situational cues such as those provided by instructions or the sex of a game partner.

Summary We report on two cases of necrotizing fasciitis of the lower leg due to nontoxigenic Vibrio cholerae (V. cholerae). A 73-year-old woman (case 1) and an 80-year-old man (case 2) were hospitalized with symptoms of necrotizing fasciitis on July 18 and August 15, 2015, respectively. In both cases, symptoms started the day after swimming in local ponds. Swabs gained intraoperatively and a blood culture from the male patient, yielded V. cholerae non-O1/non-O139, negative for cholera toxin gene ctx and positive for hemolysin genes hlyA and hlyB. Water samples taken from pond A on August 17, 2015 (32 days after exposure of case 1) and from pond B on August 20, 2015 (7 days after exposure of case 2) yielded non-O1/non-O139 V. cholerae in most-probable numbers of > 11,000 per 100 ml each. The occurrence of two cases of necrotizing fasciitis within a 1 month period related to two Austrian non-saline bathing waters, previously not known to harbor V. cholerae, is probably linked to the prevailing extreme weather conditions (heat wave, drought) this summer in Austria. While case 1 was discharged in good clinical condition after 73 days, case 2 died after four months of hospitalization. Public health authorities are challenged to assess the effects of long-term climate change on pathogen growth and survival in continental bodies of fresh water.