Functional studies with IgM and IgA immunoglobulins: binding to pIgR, FcαµR, FcµR, and CDC activities.

IgMs are the first antibodies produced by the immune system upon encounter of a possible pathogen and are one of five antibody subclasses in humans. For IgG, the most intensively studied antibody class, the N-linked glycosylation site located in the Fc-domain is directly involved in high affinity binding to the respective receptors and initiation of corresponding immune response. IgM molecules have five N-glycosylation sites and one N-glycosylation site in the J-chain, which can be incorporated in IgM or IgA molecules. There is only limited knowledge available concerning the function of these N-glycosylations in IgMs. To address this question, we produced IgM molecules lacking a particular N-glycosylation site and tested these variants as well as IgA molecules for binding to the known receptors: the polymeric immunoglobulin receptor (pIgR), the dual receptor for IgA and IgM, FcαµR, and the specific receptor for IgM, FcµR. The single glycosylation sites did not show an impact on expression and multimerization, except for variant N402Q, which could not be expressed. In SPR measurements, no major impact on the binding to the receptors by particular glycosylation sites could be detected. In cellular assays, deglycosylated variants showed some alterations in induction of CDC activity. Most strikingly, we observed also binding of IgA to the FcµR in the same affinity range as IgM, suggesting that this might have a physiological role. To further substantiate the binding of IgA to FcµR we used IgA from different origins and were able to confirm binding of IgA preparations to the FcµR.

H2-M and H2-O as Targeting Vehicles for the MHC Class II Processing Compartment Promote Antigen-Specific CD4+ T Cell Activation.

CD8 and CD4 T cell activation are both required for a strong and long-lasting T cell immune response. Endogenously expressed proteins are readily processed by the MHC class I antigen presentation pathway, enabling activation of CD8+ T cells. However, the MHC class II antigen presentation pathway, necessary for CD4+ T cell activation, is generally not sufficiently accessible to endogenously expressed proteins, limiting the efficiency of mRNA- or DNA-based vaccines. In the current study, we have evaluated the feasibility of using antigen sequences fused to sequences derived from the H2-M and H2-O proteins, two complexes known to participate in MHC class II antigen processing, for the enhancement of CD4 T-cell activation. We analyzed T cell activation after genetic immunization with mRNA-encoding fusion proteins with the model antigen ovalbumin and sequences derived from H2-M or H2-O. Our results show that H2-M- or H2-O-derived sequences robustly improve antigen-specific CD4 T-cell activation when fused to the antigen of interest and suggest that the approach could be used to improve the efficiency of mRNA- or DNA-based vaccines.

Utility of the RIG-I Agonist Triphosphate RNA for Melanoma Therapy.

The pattern recognition receptor RIG-I plays an important role in the recognition of nonself RNA and antiviral immunity. RIG-I's natural ligand, triphosphate RNA (ppp-RNA), is proposed to be a valuable addition to the growing arsenal of cancer immunotherapy treatment options. In this study, we present comprehensive data validating the concept and utility of treatment with synthetic RIG-I agonist ppp-RNA for the therapy of human cancer, with melanoma as potential entry indication amenable to intratumoral treatment. Using mRNA expression data of human tumors, we demonstrate that RIG-I expression is closely correlated to cellular and cytokine immune activation in a wide variety of tumor types. Furthermore, we confirm susceptibility of cancer cells to ppp-RNA treatment in different cellular models of human melanoma, revealing unexpected heterogeneity between cell lines in their susceptibility to RNA agonist features, including sequence, secondary structures, and presence of triphosphate. Cellular responses to RNA treatment (induction of type I IFN, FasR, MHC-I, and cytotoxicity) were demonstrated to be RIG-I dependent using KO cells. Following ppp-RNA treatment of a mouse melanoma model, we observed significant local and systemic antitumor effects and survival benefits. These were associated with type I IFN response, tumor cell apoptosis, and innate and adaptive immune cell activation. For the first time, we demonstrate systemic presence of tumor antigen-specific CTLs following treatment with RIG-I agonists. Despite potential challenges in the generation and formulation of potent RIG-I agonists, ppp-RNA or analogues thereof have the potential to play an important role for cancer treatment in the next wave of immunotherapy.

Transcriptional profiling in C. elegans suggests DNA damage dependent apoptosis as an ancient function of the p53 family.

BACKGROUND: In contrast to the three mammalian p53 family members, p53, which is generally involved in DNA damage responses, and p63 and p73 which are primarily needed for developmental regulation, cep-1 encodes for the single C. elegans p53-like gene. cep-1 acts as a transcription activator in a primordial p53 pathway that involves CEP-1 activation and the CEP-1 dependent transcriptional induction of the worm BH3 only domain encoding genes egl-1 and ced-13 to induce germ cell apoptosis. EGL-1 and CED-13 proteins inactivate Bcl-2 like CED-9 to trigger CED-4 and CED-3 caspase dependent germ cell apoptosis. To address the function of p53 in global transcriptional regulation we investigate genome-wide transcriptional responses upon DNA damage and cep-1 deficiency. RESULTS: Examining C. elegans expression profiles using whole genome Affymetrix GeneChip arrays, we found that 83 genes were induced more than two fold upon ionizing radiation (IR). None of these genes, with exception of an ATP ribosylase homolog, encode for known DNA repair genes. Using two independent cep-1 loss of function alleles we did not find genes regulated by cep-1 in the absence of IR. Among the IR-induced genes only three are dependent on cep-1, namely egl-1, ced-13 and a novel C. elegans specific gene. The majority of IR-induced genes appear to be involved in general stress responses, and qRT-PCR experiments indicate that they are mainly expressed in somatic tissues. Interestingly, we reveal an extensive overlap of gene expression changes occurring in response to DNA damage and in response to bacterial infection. Furthermore, many genes induced by IR are also transcriptionally regulated in longevity mutants suggesting that DNA damage and aging induce an overlapping stress response. CONCLUSION: We performed genome-wide gene expression analyses which indicate that only a surprisingly small number of genes are regulated by CEP-1 and that DNA damage induced apoptosis via the transcriptional induction of BH3 domain proteins is likely to be an ancient DNA damage response function of the p53 family. Interestingly, although the apoptotic response to DNA damage is regulated through the transcriptional activity of CEP-1, other DNA damage responses do not appear to be regulated on the transcriptional level and do not require the p53 like gene cep-1.