Strategies for controlling the innate immune activity of conventional and self-amplifying mRNA therapeutics: Getting the message across.

The recent approval of messenger RNA (mRNA)-based vaccines to combat the SARS-CoV-2 pandemic highlights the potential of both conventional mRNA and self-amplifying mRNA (saRNA) as a flexible immunotherapy platform to treat infectious diseases. Besides the antigen it encodes, mRNA itself has an immune-stimulating activity that can contribute to vaccine efficacy. This self-adjuvant effect, however, will interfere with mRNA translation and may influence the desired therapeutic outcome. To further exploit its potential as a versatile therapeutic platform, it will be crucial to control mRNA's innate immune-stimulating properties. In this regard, we describe the mechanisms behind the innate immune recognition of mRNA and provide an extensive overview of strategies to control its innate immune-stimulating activity. These strategies range from modifications to the mRNA backbone itself, optimization of production and purification processes to the combination with innate immune inhibitors. Furthermore, we discuss the delicate balance of the self-adjuvant effect in mRNA vaccination strategies, which can be both beneficial and detrimental to the therapeutic outcome.

Analysis in epithelial ovarian cancer identifies KANSL1 as a biomarker and target gene for immune response and HDAC inhibition.

OBJECTIVE: There is an immunoreactive subtype of ovarian cancer with a favorable prognosis, but the majority of ovarian cancers have limited immune reactivity. The reason for this is poorly understood. This study aimed to approach this question by identifying prognostically relevant genes whose prognostic mRNA expression levels correlated with a genomic event. METHODS: Expression microarray and 5-year survival data on 170 ovarian tumors and aCGH data on 45 ovarian cancer cell lines were used to identify amplified/deleted genes associated with prognosis. Three immune-response genes were identified mapping to epigenetically modified chromosome 6p21.3. Genes were searched for roles in epigenetic modification, identifying KANSL1. Genome-wide association studies were searched to identify genetic variants in KANSL1 associated with altered immune profile. Sensitivity to HDAC inhibition in cell lines with KANSL1 amplification/rearrangement was studied. RESULTS: Expression of 196 genes was statistically significantly associated with survival, and expression levels correlated with copy number variations for 82 of them. Among these, 3 immune-response genes (HCP5, PSMB8, PSMB9) clustered together at epigenetically modified chromosome 6p21.3 and their expression was inversely correlated to epigenetic modification gene KANSL1. KANSL1 is amplified/rearranged in ovarian cancer, associated with lymphocyte profile, a biomarker for response to HDAC inhibition, and may drive expression of immune-response genes. CONCLUSION: This study identifies 82 genes with prognostic relevance and genomic alteration in ovarian cancer. Among these, immune-response genes have correlated expression which is associated with 5-year survival. KANSL1 may be a master gene altering immune-response gene expression at 6p21.3 and drive response to HDAC inhibitors. Future research should investigate KANSL1 and determine whether targeting it alters the immune profile of ovarian cancer and improves survival, HDAC inhibition, and/or immunotherapy response.

Evolution of the rodent Trim5 cluster is marked by divergent paralogous expansions and independent acquisitions of TrimCyp fusions.

Evolution of cellular innate immune genes in response to viral threats represents a rich area of study for understanding complex events that shape mammalian genomes. One of these genes, TRIM5, is a retroviral restriction factor that mediates a post-entry block to infection. Previous studies on the genomic cluster that contains TRIM5 identified different patterns of gene amplification and the independent birth of CypA gene fusions in various primate species. However, the evolution of Trim5 in the largest order of mammals, Rodentia, remains poorly characterized. Here, we present an expansive phylogenetic and genomic analysis of the Trim5 cluster in rodents. Our findings reveal substantial evolutionary changes including gene amplifications, rearrangements, loss and fusion. We describe the first independent evolution of TrimCyp fusion genes in rodents. We show that the TrimCyp gene found in some Peromyscus species was acquired about 2 million years ago. When ectopically expressed, the P. maniculatus TRIMCyp shows anti-retroviral activity that is reversed by cyclosporine, but it does not activate Nf-κB or AP-1 promoters, unlike the primate TRIMCyps. These results describe a complex pattern of differential gene amplification in the Trim5 cluster of rodents and identify the first functional TrimCyp fusion gene outside of primates and tree shrews.

C/EBPß is involved in the amplification of early granulocyte precursors during candidemia-induced "emergency" granulopoiesis.

Granulopoiesis is tightly regulated to meet host demands during both "steady-state" and "emergency" situations, such as infections. The transcription factor CCAAT/enhancer binding protein ß (C/EBPß) plays critical roles in emergency granulopoiesis, but the precise developmental stages in which C/EBPß is required are unknown. In this study, a novel flow cytometric method was developed that successfully dissected mouse bone marrow cells undergoing granulopoiesis into five distinct subpopulations (#1-5) according to their levels of c-Kit and Ly-6G expression. After the induction of candidemia, rapid mobilization of mature granulocytes and an increase in early granulocyte precursors accompanied by cell cycle acceleration was followed by a gradual increase in granulocytes originating from the immature populations. Upon infection, C/EBPß was upregulated at the protein level in all the granulopoietic subpopulations. The rapid increase in immature subpopulations #1 and #2 observed in C/EBPß knockout mice at 1 d postinfection was attenuated. Candidemia-induced cell cycle acceleration and proliferation of hematopoietic stem/progenitors were also impaired. Taken together, these data suggest that C/EBPß is involved in the efficient amplification of early granulocyte precursors during candidemia-induced emergency granulopoiesis.

Transient enhanced IL-2R signaling early during priming rapidly amplifies development of functional CD8+ T effector-memory cells.

Much is known concerning the cellular and molecular basis for CD8(+) T memory immune responses. Nevertheless, conditions that selectively support memory generation have remained elusive. In this study, we show that an immunization regimen that delivers TCR signals through a defined antigenic peptide, inflammatory signals through LPS, and growth and differentiation signals through the IL-2R initially favors Ag-specific CD8(+) T cells to develop rapidly and substantially into T effector-memory cells by TCR transgenic OVA-specific OT-I CD8(+) T cells. Amplified CD8(+) T memory development depends upon a critical frequency of Ag-specific T cells and direct responsiveness to IL-2. A homologous prime-boost immunization protocol with transiently enhanced IL-2R signaling in normal mice led to persistent polyclonal Ag-specific CD8(+) T cells that supported protective immunity to Listeria monocytogenes. These results identify a general approach for amplified T memory development that may be useful to optimize vaccines aimed at generating robust cell-mediated immunity.

Mitochondrial transcription factor A serves as a danger signal by augmenting plasmacytoid dendritic cell responses to DNA.

Plasmacytoid dendritic cells (pDC) are potent APCs known to regulate immune responses to self-Ags, particularly DNA. The mitochondrial fraction of necrotic cells was found to most potently promote human pDC activation, as reflected by type I IFN release, which was dependent upon the presence of mitochondrial DNA and involved TLR9 and receptors for advanced glycation end products. Mitochondrial transcription factor A (TFAM), a highly abundant mitochondrial protein that is functionally and structurally homologous to high mobility group box protein 1, was observed to synergize with CpG-containing oligonucleotide, type A, DNA to promote human pDC activation. pDC type I IFN responses to TFAM and CpG-containing oligonucleotide, type A, DNA indicated their engagement with receptors for advanced glycation end products and TLR9, respectively, and were dependent upon endosomal processing and PI3K, ERK, and NF-κB signaling. Taken together, these results indicate that pDC contribute to sterile immune responses by recognizing the mitochondrial component of necrotic cells and further incriminate TFAM and mitochondrial DNA as likely mediators of pDC activation under these circumstances.

Antibody-mediated coengagement of FcγRIIb and B cell receptor complex suppresses humoral immunity in systemic lupus erythematosus.

Engagement of the low-affinity Ab receptor FcγRIIb downregulates B cell activation, and its dysfunction is associated with autoimmunity in mice and humans. We engineered the Fc domain of an anti-human CD19 Ab to bind FcγRIIb with high affinity, promoting the coengagement of FcγRIIb with the BCR complex. This Ab (XmAb5871) stimulated phosphorylation of the ITIM of FcγRIIb and suppressed BCR-induced calcium mobilization, proliferation, and costimulatory molecule expression of human B cells from healthy volunteers and systemic lupus erythematosus (SLE) patients, as well as B cell proliferation induced by LPS, IL-4, or BAFF. XmAb5871 suppressed humoral immunity against tetanus toxoid and reduced serum IgM, IgG, and IgE levels in SCID mice engrafted with SLE or healthy human PBMC. XmAb5871 treatment also increased survival of mice engrafted with PBMC from a unique SLE patient. Unlike anti-CD20 Ab, coengagement of FcγRIIb and BCR complex did not promote B cell depletion in human PBMC cultures or in mice. Thus, amplification of the FcγRIIb inhibitory pathway in activated B cells may represent a novel B cell-targeted immunosuppressive therapeutic approach for SLE and other autoimmune diseases that should avoid the complications associated with B cell depletion.

Regulation of the foxp3 gene by the Th1 cytokines: the role of IL-27-induced STAT1.

Impaired functional activity of T regulatory cells has been reported in allergic patients and results in an increased susceptibility to autoimmune diseases. The master regulator of T regulatory cell differentiation, the transcription factor FOXP3, is required for both their development and function. Despite its key role, relatively little is known about the molecular mechanisms regulating foxp3 gene expression. In the present study, the effect of Th1 cytokines on human T regulatory cell differentiation was analyzed at epigenetic and gene expression levels and reveals a mechanism by which the STAT1-activating cytokines IL-27 and IFN-gamma amplify TGF-beta-induced FOXP3 expression. This study shows STAT1 binding elements within the proximal part of the human FOXP3 promoter, which we previously hypothesized to function as a key regulatory unit. Direct binding of STAT1 to the FOXP3 promoter following IL-27 stimulation increases its transactivation process and induces permissive histone modifications in this key region of the FOXP3 promoter, suggesting that FOXP3 expression is promoted by IL-27 by two mechanisms. Our data demonstrate a molecular mechanism regulating FOXP3 expression, which is of considerable interest for the development of new drug targets aiming to support anti-inflammatory mechanisms of the immune system.

From gene amplification to V(D)J recombination and back: a personal account of my early years in B cell biology.

I have been invited to write a short historical feature in the context of being a co-recipient with Klaus Rajewsky and Fritz Melchers of the 2007 Novartis Prize in Basic Immunology that was given in the general area of the molecular biology of B cells. In this feature, I cover the main points of the short talk that I presented at the Award Ceremony at the International Immunology Congress in Rio de Janeiro, Brazil. This talk focused primarily on the work and people involved early on in generating the models and ideas that have formed the basis for my ongoing efforts in the areas of V(D)J recombination and B cell development.

Pathological role of osteoclast costimulation in arthritis-induced bone loss.

Abnormal T cell immune responses induce aberrant expression of inflammatory cytokines such as TNF-alpha, leading to osteoclastmediated bone erosion and osteoporosis in autoimmune arthritis. However, the mechanism underlying enhanced osteoclastogenesis in arthritis is not completely understood. Here we show that TNF-alpha contributes to inflammatory bone loss by enhancing the osteoclastogenic potential of osteoclast precursor cells through inducing paired Ig-like receptor-A (PIR-A), a costimulatory receptor for receptor activator of NF-kappaB (RANK). In fact, bone erosion and osteoporosis, but not inflammation, caused by aberrant TNF-alpha expression were ameliorated in mice deficient in Fc receptor common gamma subunit or beta(2)-microglobulin, in which the expression of PIR-As and PIR-A ligands is impaired, respectively. These results establish the pathological role of costimulatory receptors for RANK in bone loss in arthritis and may provide a molecular basis for the future therapy of inflammatory diseases.

TLR4 up-regulation at protein or gene level is pathogenic for lupus-like autoimmune disease.

TLR4 is the receptor for the Gram-negative bacterial cell wall component LPS. TLR4 signaling is controlled by both positive and negative regulators to balance optimal immune response and potential sepsis. Unchecked TLR4 activation might result in autoimmune diseases, a hypothesis that has not been formally resolved. In this study, we found that TLR4 signaling to LPS can be positively enforced by expressing gp96 on cell surfaces through the chaperone function of, but not the direct signaling by, gp96; TLR4 as well as the commensal flora are essential for the production of anti-dsDNA Ab and the immune complex-mediated glomerulonephritis in transgenic mice that express surface gp96. Moreover, a similar constellation of autoimmunity was evident in mice that encode multiple copies of tlr4 gene. Our study has revealed that increased TLR4 signaling alone without exogenous insult can break immunological tolerance. It provides a strong experimental evidence for TLR4 dysregulation as an etiology of lupus-like renal disease.

Be fruitful and multiply: gene amplification inducing pathogen resistance.

The majority of European spring barley cultivars have broad-spectrum powdery mildew resistance conditioned by a naturally occurring recessive resistance allele at the Mlo locus that originates from Ethiopian landraces. A recent publication by Pietro Piffanelli et al. describes an unusual amplification of gene segments upstream of the Mlo promoter that probably interferes with transcription and is responsible for gene inactivation. Their findings provide insights into the breeding events during the domestication of wild barley.

Downregulation of the cellular adhesion molecule Thy-1 (CD90) by cytomegalovirus infection of human fibroblasts.

The deregulation of cellular adhesion molecules by human cytomegalovirus (HCMV) appears to be correlated with the development of vascular disease. In this study, it was investigated whether the expression of Thy-1 (CD90), a member of the immunoglobulin superfamily of adhesion molecules with constitutive expression on fibroblast cells, is modulated following infection with HCMV. It was observed that Thy-1 cell surface expression decreased significantly during the course of infection. Addition of neutralizing antibodies, as well as UV inactivation of virus, prevented Thy-1 downregulation. In contrast, inhibition of virus replication by cidofovir did not alter Thy-1 regulation by HCMV, indicating that immediate-early (IE) and/or early (E) gene products are responsible. Interestingly, after infection of fibroblasts with a recombinant GFP-expressing virus, infected as well as non-infected cells showed a reduced Thy-1 cell surface expression. From these findings, it is concluded that IE or E gene products of HCMV induce a so far unidentified soluble factor that mediates Thy-1 downregulation.

c-jun mRNA expression and profilin mRNA amplification in rat alveolar macrophages exposed to volcanic ash and sulfur dioxide.

Local residents exposed to heavy falls of ash discharged by Mt. Sakurajima, an active volcano, have been reported to develop acute and chronic inflammation of the respiratory tract. The present study aimed to determine the primary cause of this inflammation using an experimental model. Wistar rats were exposed for 5 days (4 h/d) to air containing 100 mg/m3 volcanic ash (mass median aerodynamic diameter, 4.3 microm; geometric standard deviation, 1.7) with or without 1.5 ppm sulfur dioxide (SO2). The lungs were then lavaged, and mRNA was extracted from alveolar macrophages and assessed by reverse transcription-polymerase chain reaction (RT-PCR). In the lavage fluid, no change in cellularity or increase in the content of tumor necrosis factor (TNF)-alpha was detected. However, at 1 h following exposure, 80% of macrophages were seen to have phagocytosed the volcanic ash. This percentage was unchanged at 24 h after exposure. Profilin mRNA content of the macrophages was elevated, and c-jun mRNA was expressed. Alveolar macrophages exposed to volcanic ash and SO2, therefore, are likely to have some inflammatory and fibrogenic potential.

Killer immunoglobulin-like receptor expression on single cells: a cautionary note.

Natural killer (NK) cells keep the surface expression of major histocompatibility complex (MHC) class I molecules under surveillance using killer immunoglobulin-like receptors (KIR). Virus-infected or aberrant cells are frequently characterized by a reduced surface expression of MHC class I antigens and may therefore be removed by cytolysis. NK cells are heterogeneous with regard to the expression of KIR genes. The resulting subpopulations show distinguishable specificities allowing the recognition of cells lacking varying combinations of MHC class I antigens. The KIR expression pattern in single NK cells has previously been analyzed by Husain and colleagues by cDNA preamplification of CD3- CD56+ single cells and subsequent gene-specific polymerase chain reaction. We show here that the data of this study contain inconsistencies. These inconsistencies are discussed in the context of KIR mRNA abundance and single-cell cDNA amplification efficiency.

Unraveling of the polymorphic C lambda 2-C lambda 3 amplification and the Ke+Oz- polymorphism in the human Ig lambda locus.

Two polymorphisms of the human Ig(lambda) (IGL) locus have been described. The first polymorphism concerns a single, 2- or 3-fold amplification of 5.4 kb of DNA in the C(lambda)2-C(lambda)3 region. The second polymorphism is the Mcg(-)Ke(+)Oz(-) isotype, which has only been defined via serological analyses in Bence-Jones proteins of multiple myeloma patients and was assumed to be encoded by a polymorphic C(lambda)2 segment because of its high homology with the Mcg(-)Ke(-)Oz(-) C(lambda)2 isotype. It has been speculated that the Mcg(-)Ke(+)Oz(-) isotype might be encoded by a C(lambda) gene segment of the amplified C(lambda)2-C(lambda)3 region. We now unraveled both IGL gene polymorphisms. The amplification polymorphism appeared to result from a duplication, triplication, or quadruplication of a functional J-C(lambda)2 region and is likely to have originated from unequal crossing over of the J-C(lambda)2 and J-C(lambda)3 region via a 2.2-kb homologous repeat. The amplification polymorphism was found to result in the presence of one to five extra functional J-C(lambda)2 per genome regions, leading to decreased Ig(kappa):Ig(lambda) ratios on normal peripheral blood B cells. Via sequence analysis, we demonstrated that the Mcg(-)Ke(+)Oz(-) isotype is encoded by a polymorphic C(lambda)2 segment that differs from the normal C(lambda)2 gene segment at a single nucleotide position. This polymorphism was identified in only 1.5% (2 of 134) of individuals without J-C(lambda)2 amplification polymorphism and was not found in the J-C(lambda)2 amplification polymorphism of 44 individuals, indicating that the two IGL gene polymorphisms are not linked.

Minimal requirements for IgE-mediated regulation of surface Fc epsilon RI.

The IgE-FcepsilonRI network plays a central role in allergic inflammation. IgE levels control cell surface levels of FcepsilonRI and, in turn, FcepsilonRI levels modulate the intensity of effector responses. Treatment of allergic patients with anti-IgE Abs has been shown to induce a decrease in FcepsilonRI expression on basophils and a decrease in Ag-triggered histamine release. However, the mechanisms underlying IgE-mediated regulation of FcepsilonRI expression remain unclear. Here, we designed an in vitro model system to establish the minimal cellular requirements for regulation of FcepsilonRI by IgE. Using this system, we demonstrate that transcriptional regulation, hemopoietic-specific factors, and signaling are not required for IgE-mediated increases in FcepsilonRI expression. IgE binding to the alpha-chain is the minimal requirement for the induction of FcepsilonRI up-regulation. The rate of up-regulation is independent of the baseline level of expression. The mechanism of this up-regulation is the result of a combination of three factors: 1) stabilization of the receptor at the cell surface, which prevents receptor internalization and degradation; 2) use of a preformed pool of receptor comprising recycled and recently synthesized receptors; and 3) continued basal level of protein synthesis. It is possible that in vivo additional factors contribute to modulate the basic regulatory mechanism described here.

Induction of polyclonal prostate cancer-specific CTL using dendritic cells transfected with amplified tumor RNA.

Polyvalent cancer vaccines targeting the entire antigenic spectrum on tumor cells may represent a superior therapeutic strategy for cancer patients than vaccines solely directed against single Ags. In this study, we show that autologous dendritic cells (DC) transfected with RNA amplified from microdissected tumor cells are capable of stimulating CTL against a broad set of unidentified and critical prostate-specific Ags. Although the polyclonal CTL responses generated with amplified tumor RNA-transfected DC encompassed as a subcomponent a response against prostate-specific Ag (PSA) as well as against telomerase reverse transcriptase, the tumor-specific CTL were consistently more effective than PSA or telomerase reverse transcriptase CTL to lyse tumor targets, suggesting the superiority of the polyclonal response. Although tumor RNA-transfected DC stimulated CTL, which recognized not only tumor but also self-Ags expressed by benign prostate tissue, these cross-reactive CTL were exclusively specific for the PSA, indicating an immunodominant role of PSA in the prostate cancer-specific immune response. Our data suggest that tumor RNA-transfected DC may represent a broadly applicable, potentially clinically effective vaccine strategy for prostate cancer patients, which is not limited by tumor tissue availability for Ag preparation and may minimize the risk of clonal tumor escape.

Amplification of IL-1 beta-induced matrix metalloproteinase-9 expression by superoxide in rat glomerular mesangial cells is mediated by increased activities of NF-kappa B and activating protein-1 and involves activation of the mitogen-activated protein kinase pathways.

The modulation of cell signaling by free radicals is important for the pathogenesis of inflammatory diseases. Recently, we have shown that NO reduces IL-1beta-induced matrix metalloproteinase (MMP-9) expression in glomerular mesangial cells (MC). Here we report that exogenously administrated superoxide, generated by the hypoxanthine/xanthine oxidase system (HXXO) or by the redox cycler 2, 3-dimethoxy-1,4-naphtoquinone, caused a marked amplification of IL-1beta-primed, steady state, MMP-9 mRNA level and an increase in gelatinolytic activity in the conditioned medium. Superoxide generators alone were ineffective. Cytokine-induced steady state mRNA levels of TIMP-1, an endogenous inhibitor of MMP-9, were affected similarly by HXXO. Transient transfection of rat mesangial cells with 0.6 kb of the 5'-flanking region of the rat MMP-9 gene proved a transcriptional regulation of MMP-9 expression by superoxide. HXXO augmented the IL-1beta-triggered nuclear translocation of p65 and c-Jun and, in parallel, increased DNA binding activities of NF-kappaB and AP-1. Mutation of either response element completely prevented MMP-9 promoter activation by IL-1beta. Moreover, specific inhibitors of the classical extracellular signal-regulated kinase (ERK) pathway and p38 mitogen-activated protein kinase (MAPK) cascade, partially reversed the HXXO-mediated effects on MMP-9 mRNA levels, thus demonstrating involvement of ERKs and p38 MAPKs in MMP-9 expression. Furthermore, IL-1beta-triggered phosphorylation of all three MAPKs, including p38-MAPK, c-Jun N-terminal kinase, and ERK, was substantially enhanced by superoxide. Our data identify superoxide as a costimulatory factor amplifying cytokine-induced MMP-9 expression by interfering with the signaling cascades leading to the activation of AP-1 and NF-kappaB.

Allergy-associated polymorphisms of the Fc epsilon RI beta subunit do not impact its two amplification functions.

Two variants of the beta-chain of the high affinity IgE receptor Fc epsilon RI, I181L-V183L and E237G, have been found associated with allergy. We have previously shown that the beta-chain plays at least two distinct amplifier functions. It amplifies Fc epsilon RI surface expression and signaling, resulting in an estimated 12- to 30-fold amplification of downstream events. To test the hypothesis that the I181L-V183L and E237G beta variants may be functionally relevant and could directly contribute to an allergic phenotype, we have evaluated the functional impact of the beta variants on the two amplifier functions of beta. We found that these variants have no direct effect on the beta amplifier functions. However, the possibility remains that these variants are in linkage disequilibrium with other more relevant polymorphisms or are affecting unknown beta-chain functions.