Targeting the Wnt signaling pathway through R-spondin 3 identifies an anti-fibrosis treatment strategy for multiple organs.

The Wnt/ß-catenin signaling pathway has been implicated in human proliferative diseases such as cancer and fibrosis. The functions of ß-catenin and several other components of this pathway have been investigated in fibrosis. However, the potential role of R-spondin proteins (RSPOs), enhancers of the Wnt/ß-catenin signaling, has not been described. A specific interventional strategy targeting this pathway for fibrosis remains to be defined. We developed monoclonal antibodies against members of the RSPO family (RSPO1, 2, and 3) and probed their potential function in fibrosis in vivo. We demonstrated that RSPO3 plays a critical role in the development of fibrosis in multiple organs. Specifically, an anti-RSPO3 antibody, OMP-131R10, when dosed therapeutically, attenuated fibrosis in carbon tetrachloride (CCl4)-induced liver fibrosis, bleomycin-induced pulmonary and skin fibrosis models. Mechanistically, we showed that RSPO3 induces multiple pro-fibrotic chemokines and cytokines in Kupffer cells and hepatocytes. We found that the anti-fibrotic activity of OMP-131R10 is associated with its inhibition of ß-catenin activation in vivo. Finally, RSPO3 was found to be highly elevated in the active lesions of fibrotic tissues in mouse models of fibrosis and in patients with idiopathic pulmonary fibrosis (IPF) and nonalcoholic steatohepatitis (NASH). Together these data provide an anti-fibrotic strategy for targeting the Wnt/ß-catenin pathway through RSPO3 blockade and support that OMP-131R10 could be an important therapeutic agent for fibrosis.

An activin receptor IIA ligand trap promotes erythropoiesis resulting in a rapid induction of red blood cells and haemoglobin.

Sotatercept (ACE-011), a recombinant human fusion protein containing the extracellular domain of the human Activin receptor IIA, binds to and inhibits activin and other members of the transforming growth factor -ß (TGF-ß) superfamily. Administration of sotatercept led to a rapid and sustained increase in red blood cell (RBC) count and haemoglobin (Hb) in healthy volunteers (phase I clinical trials), but the mechanism is not fully understood. Mice treated with RAP-011 (murine ortholog of ACE-011) respond with a rapid (within 24 h) increase in haematocrit, Hb, and RBC count. These effects are accompanied by an equally rapid stimulation of late-stage erythroid precursors in the bone marrow (BM). RAP-011 also induces a significant increase in erythroid burst-forming units and erythropoietin, which could contribute to additional, sustained effects on RBC production. Further in vitro co-culture studies demonstrate that BM accessory cells are required for RAP-011 effects. To better understand which TGF-ß family ligand(s) mediate RAP-011 effects, we evaluated the impact of several of these ligands on erythroid differentiation. Our data suggest that RAP-011 may act to rescue growth differentiation factor 11/Activin A-induced inhibition of late-stage erythropoiesis. These data define the mechanism of action of a novel agent that regulates RBC differentiation and provide the rationale to develop sotatercept for the treatment of anaemia and ineffective erythropoiesis.

Optimized clinical performance of growth hormone with an expanded genetic code.

The ribosomal incorporation of nonnative amino acids into polypeptides in living cells provides the opportunity to endow therapeutic proteins with unique pharmacological properties. We report here the first clinical study of a biosynthetic protein produced using an expanded genetic code. Incorporation of p-acetylphenylalanine (pAcF) at distinct locations in human growth hormone (hGH) allowed site-specific conjugation with polyethylene glycol (PEG) to produce homogeneous hGH variants. A mono-PEGylated mutant hGH modified at residue 35 demonstrated favorable pharmacodynamic properties in GH-deficient rats. Clinical studies in GH-deficient adults demonstrated efficacy and safety comparable to native human growth hormone therapy but with increased potency and reduced injection frequency. This example illustrates the utility of nonnative amino acids to optimize protein therapeutics in an analogous fashion to the use of medicinal chemistry to optimize conventional natural products, low molecular weight drugs, and peptides.

Principles of memory CD8 T-cells generation in relation to protective immunity.

Memory T-cell responses are of vital importance in understanding the host's response against pathogens and cancer cells and to begin establishing the correlation of protection against disease. In this review, we discuss our own data in the general context of current knowledge to sketch tentative working principles for the induction of protective T-cell responses by vaccination. We draw attention to quantitative and qualitative aspects of the initial contact with antigen, as well as to the kinetics of events leading to the generation of memory T cells thereafter. Our arguments are based on the current distinction of memory T cells into two lineages: effector memory T cells (T(EM)) and central memory T cells (T(CM)). Our provisional conclusion is that protective T-cell responses correlate positively with the T cells of the central memory phenotype. In proposing a set of working principles to enable protective memory T cells by vaccination we address vaccination both in the context of the immunologically-inexperienced and immunologically-experienced individual, respectively. Finally, we draw attention to the interplay between systemic and local immunity as important factors in determining the success of memory T-cell responses in protecting the individual. We believe that considerations on the immunodynamics of memory induction and maintenance, memory lineage differentiation and their relation to protection may help design strategies to control disease caused by pathogens and cancer.

Protection against influenza A virus by memory CD8 T cells requires reactivation by bone marrow-derived dendritic cells.

Influenza A virus is the causative agent of an acute inflammatory disease of the airway. Although Abs can prevent infection, disease and death can be prevented by T cell-mediated immunity. Recently, we showed that protection against lethal influenza A (PR8/34) virus infection is mediated by central memory CD8 T cells (T(CM)). In this study, using relB(-/-) mice we began to investigate the role of bone marrow (BM)-derived dendritic cells (DCs) in the mechanism of protection. We found that in the absence of functional DCs, memory CD8 T cells specific for the nucleoprotein epitope (NP(366-374)) fail to protect even after adoptive transfer into naive recipients. Through an analysis of Ag uptake, activation of memory CD8 T cells, and display of peptide/MHC complex by DCs in draining LNs and spleen early after virus infection, we established that lack of protection is associated with defective Ag presentation by BM-derived DCs and defective homing of memory T cells in the lymph nodes draining the airway tract. Collectively, the data suggest that protection against the influenza A virus requires that memory CD8 T cells be reactivated by Ag presented by BM-derived DCs in the lymph nodes draining the site of infection. They also imply that protection depends both on the characteristics of systemic adaptive immunity and on the coordinated interplay between systemic and local immunity.

TLR9-independent activation of B lymphocytes by bacterial DNA.

The intracellular Toll-like receptor 9 (TLR9) is unique in its ability to recognize single-stranded DNA unmethylated at CpG motifs. Work from this laboratory showed that plasmid DNA is spontaneously internalized in B lymphocytes. This event is followed by the upregulation of costimulatory molecules and the acquisition of antigen presenting function by these cells. However, it is not known whether this phenomenon depends on TLR9. Because of the relevant role played by DNA-based drugs in immunotherapy and vaccination, and the central role of TLR9 signaling by CpG motifs, we decided to investigate whether signaling through TLR9 is a prerequisite for spontaneous transgenesis of lymphocytes. Here we found that transgene expression and upregulation of CD40 and CD86 costimulatory molecules was not inhibited by chloroquine treatment. Spontaneous transgenesis also occurred in B lymphocytes from TLR9-/- mice, and the injection of TLR9-/- transgenic B lymphocytes in C57Bl/6 mice induced both CD4 and CD8 T cell responses comparable to those induced by wild-type B lymphocytes. Collectively, these results suggest that plasmid DNA activates mammalian B lymphocytes through a TLR9 independent pathway.

The cooperation between two CD4 T cells induces tumor protective immunity in MUC.1 transgenic mice.

Immunity and tumor protection in mice transgenic for human MUC.1, a glycoprotein expressed in the majority of cancers of epithelial origin in humans, were induced by vaccination with B lymphocytes genetically programmed to activate MUC.1-specific CD4 T cells. Their activation required a functional cooperation between two Th cells, one specific for a self (MUC.1) and the other for a nonself T cell determinant. The immunological switch provided by Th-Th cooperation was sufficient to induce MUC.1-specific CD4 and CD8 T cell responses in MUC.1-transgenic mice, and protect them permanently from tumor growth. CD4 T cells specific for MUC.1 lacked cytolytic function, but produced IFN-gamma upon restimulation with Ag. We conclude that immunity against tumor self-Ags and tumor protection can be regulated exploiting an inherent property of the immune system.

CD8 T cell priming by B lymphocytes is CD4 help dependent.

While it is generally accepted that B lymphocytes can present antigen and activate CD4 T cells, priming of CD8 T cells by B lymphocytes remains controversial. Recently, we showed that mice injected with genetically programmed B lymphocytes generate antigen specific CD4 and CD8 T cell responses in vivo that could also be induced in mice lacking functional dendritic cells. To gain further insights into the requirements for T cell priming by antigen-presenting B lymphocytes, in vitro experiments were performed using ovalbumin (OVA) and OVA-specific TCR-transgenic CD4 and CD8 T cells. We found that while B lymphocytes can directly prime CD4 T cells, the activation of CD8 T cells requires T cell help. Transfer experiments show that help can either be contact dependent or be mediated by soluble factors in the supernatants of activated OVA-specific CD4 T cells. Furthermore, the effect of activated CD4 T cells can be replaced by soluble recombinant IL-4. Collectively, the data show the existence of different requirements for priming of CD4 and CD8 T cells and point to the previously unappreciated fact that the induction of CD8 T cell responses by B lymphocytes requires T cell help.

Genetically programmed B lymphocytes are highly efficient in inducing anti-virus protective immunity mediated by central memory CD8 T cells.

The hallmarks of specific T cell immunity include proliferative expansion, acquisition of effector function and memory T cell formation. Here, we used priming with B lymphocytes transgenic for the dominant epitope (NP366-374) of the influenza virus nucleoprotein, to study the characteristics of the CD8 T cell memory response in C57Bl/6 mice and elucidate which subset of CD8 T cells memory mediates protection from disease. We found that (i) the size of the memory CTL response is independent of the priming dose and is similar to that induced by the live virus, (ii) priming with a low dose (3 x 10(2)cells/inoculum) of transgenic B lymphocytes confers a protective memory CTL response, and (iii) protection from disease is mediated by central memory (T(CM)) CD8 T cells.

B lymphocytes as antigen-presenting cell-based genetic vaccines.

Inoculation of plasmid DNA is a simple way to immunize, but it is characterized by low immunogenicity, which has hampered the development of effective DNA vaccines for human use. Here, we discuss how poor immunogenicity can be solved and present our proposal: genetically programmed B lymphocytes as antigen-presenting cell (APC) vaccines. First, we demonstrate that mature B lymphocytes take up plasmid DNA spontaneously, i.e., in the absence of any facilitating molecule or event, spontaneous lymphocyte transgenesis. Second, we demonstrate that transgenic B lymphocytes are easily and reproducibly turned into functional APCs with dual characteristics: upregulation of costimulatory molecules and endogenous synthesis of antigen. Used as immunogens in mice, transgenic B lymphocytes induce robust and long-lasting T-cell immunity after single intravenous injection. Surprisingly, immunity and protection against lethal virus challenge can be obtained with a single intravenous injection of 3 x 10(2) transgenic lymphocytes. The new approach is discussed relative to the advantage of targeting secondary lymphoid organs with genetically programmed B lymphocytes and the advantage offered with respect to low antigen dose. We suggest that these properties reflect on simple characteristics, such as time synchronization and initial localization to secondary lymphoid organs of APCs endowed with protracted synthesis and presentation of antigen to T cells.

T cell immunity using transgenic B lymphocytes.

Adaptive immunity exists in all vertebrates and plays a defense role against microbial pathogens and tumors. T cell responses begin when precursor T cells recognize antigen on specialized antigen-presenting cells and differentiate into effector cells. Currently, dendritic cells are considered the only cells capable of stimulating T lymphocytes. Here, we show that mature naïve B lymphocytes can be genetically programmed by using nonviral DNA and turned into powerful antigen-presenting cells with a dual capacity of synthesis and presentation of antigen to T cells in vivo. A single i.v. injection of transgenic lymphocytes activates T cell responses reproducibly and specifically even at very low cell doses (approximately 10(2)). We also demonstrate that T cell priming can occur in the absence of dendritic cells and results in immunological memory with protective effector functions. These findings disclose aspects in the regulation of adaptive immunity and indicate possibilities for vaccination against viruses and cancer in humans.

The role of relB in regulating the adaptive immune response.

Dendritic cells (DCs), which represent a key type of antigen-presenting cell (APC), are important for the development of innate and adaptive immunity. DCs are involved in T cell activation in at least two main ways: priming via direct processing/presentation of soluble antigen taken up from the microenvironment (conventional priming), and processing/presentation of antigen released from other cells (cross-priming). relB, a component of the NF-kappaB complex of transcription factors, is a critical regulator of the differentiation of DCs. In mice, lack of relB impairs DCs derived from bone marrow both in number and function. Here relB (-/-) bone marrow chimera mice is used to study the APC function of residual DCs in presentation of soluble antigen and cross-priming. It is found that the DCs in these mice are profoundly deficient in their ability to both prime and cross-prime T cell responses. It was concluded that the relB gene is involved in regulating the APC function of DCs in vivo.

Role of T cell help and endoplasmic reticulum targeting in protective CTL response against influenza virus.

We report on the induction of primary and long-term memory cytotoxic T lymphocyte (CTL) responses against the nucleoprotein of the influenza virus A/PR8/34 in mice immunized with plasmid DNA targeted to B lymphocytes in the spleen. We found that the magnitude of the CTL response and the size of the pool of memory CTL was greater when the CTL response was induced in presence of T cell help. Interestingly, immunization with a signal sequence-competent transgene was markedly superior to immunization with a transgene lacking the endoplasmic reticulum (ER) targeting sequence, in inducing CTL. We also found a correlation between in vivo protection from lethal virus challenge and (1) the availability of T cell help and (2) ER targeting. Immunization of dendritic cell-deficient mice suggests that B lymphocytes function as antigen-presenting cells in this model of immunization. Collectively, the results suggest that somatic transgene immunization is a conceptually new approach to induce effective anti-viral CTL responses and to assess the parameters critical for long-lasting and protective CTL responses in vivo.

CD4 T cell priming in dendritic cell-deficient mice.

Bone marrow (BM) chimeras (BMC) generated from mice carrying a null (-/-) mutation in the relB gene of the NF-kappaB family represent an ideal model for in vivo studies on the role of dendritic cells (DC) in the adaptive immune response. The spleen and lymph nodes (LN) of relB(-/-) BMC contain a small number of residual DC, mainly CD8alpha(+), that fail to up-regulate MHC class II and co-stimulatory molecules after stimulation in vitro. Moreover, residual spleen DC of relB(-/-) BMC have a 4-fold decrease in the ability to uptake and process soluble model antigen, ovalbumin (OVA), and failed to prime CD4 and CD8 T cells in vitro and in vivo. In addition, they also failed to present OVA peptide to OT-II transgenic T lymphocytes at a normal 1:10 (stimulator:responder) cell ratio. In spite of these multiple DC defects, relB(-/-) BMC immunized with plasmid DNA targeted to the spleen as the site of immune induction develop a specific CD4(+) T cell response comparable to that of relB competent mice. These data demonstrate that CD4( +) T cells can be primed in the absence of functional DC and suggest that relB may gauge the T cell response in vivo.

Apoptosis-dependent subversion of the T-lymphocyte epitope hierarchy in lymphoma cells.

Tumor cells undergoing programmed death are an attractive source of tumor-associated antigens, and evidences are available for their therapeutic efficacy in vivo when used either alone or in association with dendritic cells. However, little is known about the specificity of the immune response induced by such antigen formulation. Indeed, activation of specific proteases during apoptosis may influence the cytoplasmic degradation of proteins and the generation of CTL epitopes. We show here that on injection of C57BL/6 mice either with RMA lymphoma cells induced to apoptosis or bone marrow-derived dendritic cells pulsed with apoptotic RMA cells, a specific and protective CTL response is induced, which, however, is not directed against the immunodominant CTL epitope gag(85-93). Lack of in vivo expansion of gag(85-93)-specific CTL in vaccinated mice is attributable to the apoptosis-dependent loss of gag(85-93) in dying tumor cells. Indeed, we found loss of gag(85-93) in RMA, MBL-2, and EL-4G+ lymphoma cells, which share gag(85-93) as an immunodominant CTL epitope, induced to apoptosis by UV irradiation, mitomycin C, doxorubicin, or daunorubicin. This phenomenon appears to be caspase-dependent, because caspase inhibition by N-benzyloxycarbonyl-Val-Ala-asp-fluoromethylketone prevents apoptosis of lymphoma cells and loss of gag(85-93). Therefore, subversion of the epitope hierarchy in apoptotic tumor cells might be relevant in the induction of tumor-specific T-lymphocyte responses.