Selective activation and expansion of regulatory T cells using lipid encapsulated mRNA encoding a long-acting IL-2 mutein.

Interleukin-2 (IL-2) is critical for regulatory T cell (Treg) function and homeostasis. At low doses, IL-2 can suppress immune pathologies by expanding Tregs that constitutively express the high affinity IL-2Rα subunit. However, even low dose IL-2, signaling through the IL2-Rß/γ complex, may lead to the activation of proinflammatory, non-Treg T cells, so improving specificity toward Tregs may be desirable. Here we use messenger RNAs (mRNA) to encode a half-life-extended human IL-2 mutein (HSA-IL2m) with mutations promoting reliance on IL-2Rα. Our data show that IL-2 mutein subcutaneous delivery as lipid-encapsulated mRNA nanoparticles selectively activates and expands Tregs in mice and non-human primates, and also reduces disease severity in mouse models of acute graft versus host disease and experimental autoimmune encephalomyelitis. Single cell RNA-sequencing of mouse splenic CD4+ T cells identifies multiple Treg states with distinct response dynamics following IL-2 mutein treatment. Our results thus demonstrate the potential of mRNA-encoded HSA-IL2m immunotherapy to treat autoimmune diseases.

Granulocyte-macrophage colony-stimulating factor mRNA and Neuroprotective Immunity in Parkinson's disease.

Restoring numbers and function of regulatory T cells (Tregs) is a novel therapeutic strategy for neurodegenerative disorders. Whether Treg function is boosted by adoptive cell transfer, pharmaceuticals, or immune modulators, the final result is a robust anti-inflammatory and neuronal sparing response. Herein, a newly developed lipid nanoparticle (LNP) containing mRNA encoding granulocyte-macrophage colony-stimulating factor (Gm-csf mRNA) was developed to peripherally induce Tregs and used for treatment in preclinical Parkinson's disease (PD) models. Administration of Gm-csf mRNA to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice and rats overexpressing alpha-synuclein produced dose-dependent increases in plasma GM-CSF levels and peripheral CD4+CD25+FoxP3+ Treg populations. This upregulation paralleled nigrostriatal neuroprotection, upregulated immunosuppression-associated mRNAs that led to the detection of a treatment-induced CD4+ T cell population, and decreased reactive microgliosis. The current findings strengthen prior works utilizing immune modulation by harnessing Gm-csf mRNA to augment adaptive immune function by employing a new delivery platform to treat PD and potentially other neurodegenerative disorders.

mRNA-encoded, constitutively active STINGV155M is a potent genetic adjuvant of antigen-specific CD8+ T cell response.

mRNA vaccines induce potent immune responses in preclinical models and clinical studies. Adjuvants are used to stimulate specific components of the immune system to increase immunogenicity of vaccines. We utilized a constitutively active mutation (V155M) of the stimulator of interferon (IFN) genes (STING), which had been described in a patient with STING-associated vasculopathy with onset in infancy (SAVI), to act as a genetic adjuvant for use with our lipid nanoparticle (LNP)-encapsulated mRNA vaccines. mRNA-encoded constitutively active STINGV155M was most effective at maximizing CD8+ T cell responses at an antigen/adjuvant mass ratio of 5:1. STINGV155M appears to enhance development of antigen-specific T cells by activating type I IFN responses via the nuclear factor κB (NF-κB) and IFN-stimulated response element (ISRE) pathways. mRNA-encoded STINGV155M increased the efficacy of mRNA vaccines encoding the E6 and E7 oncoproteins of human papillomavirus (HPV), leading to reduced HPV+ TC-1 tumor growth and prolonged survival in vaccinated mice. This proof-of-concept study demonstrated the utility of an mRNA-encoded genetic adjuvant.

From the draining lymph node to the liver: the induction and effector mechanisms of malaria-specific CD8+ T cells.

Parasitic protozoa cause considerable disease in humans and, due to their intracellular life cycle, induce robust CD8(+) T cell responses. A greater understanding of the factors that promote and maintain CD8(+) T cell-mediated immunity against these pathogens is likely needed for the development of effective vaccines. Immunization with radiation-attenuated sporozoites, the infectious stage of the malaria parasite transmitted by mosquitoes, is an excellent model to study these questions as CD8(+) T cells specific for a single epitope can completely eliminate parasite infection in the liver. Furthermore, live, radiation-attenuated parasites represent the "gold standard" for malaria vaccination. Here, we will highlight recent studies aimed at understanding the factors required for the induction, recruitment, and maintenance of effector and memory CD8(+) T cells against malaria liver stages.

Proteolytic Cleavage of the Plasmodium falciparum Circumsporozoite Protein Is a Target of Protective Antibodies.

Studies in animals and human volunteers demonstrate that antibodies against the repeat-region of the Plasmodium circumsporozoite protein (CSP) abrogate sporozoite infection. However, the realization that the N- and C- terminal regions flanking the repeats play essential roles in parasite infectivity raised the possibility that they could be targeted by protective antibodies. We characterized a monoclonal antibody (mAb5D5) specific for the N-terminus of the P. falciparum CSP, which inhibits the proteolytic cleavage of the CSP, a key requirement for parasite infection of hepatocytes. Adoptive transfer of mAb5D5 strongly inhibits the in vivo infection of sporozoites expressing the N-terminus of P. falciparum CSP, and this protection is greatly enhanced when combined with antirepeat antibodies. Our results show that antibodies interfering with molecular processes required for parasite infectivity can exert a strong in vivo protective activity and indicate that pre-erythrocytic vaccines against Plasmodium should include the CSP N-terminal region.

Lymph-node resident CD8α+ dendritic cells capture antigens from migratory malaria sporozoites and induce CD8+ T cell responses.

Malaria infection begins when a female Anopheles mosquito injects Plasmodium sporozoites into the skin of its host during blood feeding. Skin-deposited sporozoites may enter the bloodstream and infect the liver, reside and develop in the skin, or migrate to the draining lymph nodes (DLNs). Importantly, the DLN is where protective CD8(+) T cell responses against malaria liver stages are induced after a dermal route of infection. However, the significance of parasites in the skin and DLN to CD8(+) T cell activation is largely unknown. In this study, we used genetically modified parasites, as well as antibody-mediated immobilization of sporozoites, to determine that active sporozoite migration to the DLNs is required for robust CD8(+) T cell responses. Through dynamic in vivo and static imaging, we show the direct uptake of parasites by lymph-node resident DCs followed by CD8(+) T cell-DC cluster formation, a surrogate for antigen presentation, in the DLNs. A few hours after sporozoite arrival to the DLNs, CD8(+) T cells are primed by resident CD8α(+) DCs with no apparent role for skin-derived DCs. Together, these results establish a critical role for lymph node resident CD8α(+) DCs in CD8(+) T cell priming to sporozoite antigens while emphasizing a requirement for motile sporozoites in the induction of CD8(+) T cell-mediated immunity.

Classical Flt3L-dependent dendritic cells control immunity to protein vaccine.

DCs are critical for initiating immunity. The current paradigm in vaccine biology is that DCs migrating from peripheral tissue and classical lymphoid-resident DCs (cDCs) cooperate in the draining LNs to initiate priming and proliferation of T cells. Here, we observe subcutaneous immunity is Fms-like tyrosine kinase 3 ligand (Flt3L) dependent. Flt3L is rapidly secreted after immunization; Flt3 deletion reduces T cell responses by 50%. Flt3L enhances global T cell and humoral immunity as well as both the numbers and antigen capture capacity of migratory DCs (migDCs) and LN-resident cDCs. Surprisingly, however, we find immunity is controlled by cDCs and actively tempered in vivo by migDCs. Deletion of Langerin(+) DC or blockade of DC migration improves immunity. Consistent with an immune-regulatory role, transcriptomic analyses reveals different skin migDC subsets in both mouse and human cluster together, and share immune-suppressing gene expression and regulatory pathways. These data reveal that protective immunity to protein vaccines is controlled by Flt3L-dependent, LN-resident cDCs.

The chemokine receptor CXCR6 is required for the maintenance of liver memory CD8⁺ T cells specific for infectious pathogens.

It is well established that immunization with attenuated malaria sporozoites induces CD8(+) T cells that eliminate parasite-infected hepatocytes. Liver memory CD8(+) T cells induced by immunization with parasites undergo a unique differentiation program and have enhanced expression of CXCR6. Following immunization with malaria parasites, CXCR6-deficient memory CD8(+) T cells recovered from the liver display altered cell-surface expression markers as compared to their wild-type counterparts, but they exhibit normal cytokine secretion and expression of cytotoxic mediators on a per-cell basis. Most importantly, CXCR6-deficient CD8(+) T cells migrate to the liver normally after immunization with Plasmodium sporozoites or vaccinia virus, but a few weeks later their numbers severely decrease in this organ, losing their capacity to inhibit malaria parasite development in the liver. These studies are the first to show that CXCR6 is critical for the development and maintenance of protective memory CD8(+) T cells in the liver.

CD8+ T cells eliminate liver-stage Plasmodium berghei parasites without detectable bystander effect.

Immunization with attenuated Plasmodium sporozoites or viral vectored vaccines can induce protective CD8(+) T cells that can find and eliminate liver-stage malaria parasites. A key question is whether CD8(+) T cells must recognize and eliminate each parasite in the liver or whether bystander killing can occur. To test this, we transferred antigen-specific effector CD8(+) T cells to mice that were then coinfected with two Plasmodium berghei strains, only one of which could be recognized directly by the transferred T cells. We found that the noncognate parasites developed normally in these mice, demonstrating that bystander killing of parasites does not occur during the CD8(+) T cell response to malaria parasites. Rather, elimination of infected parasites is likely mediated by direct recognition of infected hepatocytes by antigen-specific CD8(+) T cells.

Flt3L dependence helps define an uncharacterized subset of murine cutaneous dendritic cells.

Skin-derived dendritic cells (DCs) are potent antigen-presenting cells with critical roles in both adaptive immunity and tolerance to self. Skin DCs carry antigens and constitutively migrate to the skin-draining lymph nodes (LNs). In mice, Langerin-CD11b- dermal DCs are a low-frequency, heterogeneous, migratory DC subset that traffics to LNs (Langerin-CD11b- migDCs). Here, we build on the observation that Langerin-CD11b- migDCs are Fms-like tyrosine kinase 3 ligand (Flt3L) dependent and strongly Flt3L responsive, which may relate them to classical DCs. Examination of DC capture of FITC from painted skin, DC isolation from skin explant culture, and from the skin of CCR7 knockout mice, which accumulate migDCs, demonstrate these cells are cutaneous residents. Langerin-CD11b- Flt3L-responsive DCs are largely CD24(+) and CX3CR1(low) and can be depleted from Zbtb46-DTR mice, suggesting classical DC lineage. Langerin-CD11b- migDCs present antigen with equal efficiency to other DC subsets ex vivo, including classical CD8α cDCs and Langerin+CD103+ dermal DCs. Finally, transcriptome analysis suggests a close relationship with other skin DCs, and a lineage relationship with other classical DCs. This work demonstrates that Langerin- CD11b- dermal DCs, a previously overlooked cell subset, may be an important contributor to the cutaneous immune environment.

In vivo imaging of CD8+ T cell-mediated elimination of malaria liver stages.

CD8(+) T cells are specialized cells of the adaptive immune system capable of finding and eliminating pathogen-infected cells. To date it has not been possible to observe the destruction of any pathogen by CD8(+) T cells in vivo. Here we demonstrate a technique for imaging the killing of liver-stage malaria parasites by CD8(+) T cells bearing a transgenic T cell receptor specific for a parasite epitope. We report several features that have not been described by in vitro analysis of the process, chiefly the formation of large clusters of effector CD8(+) T cells around infected hepatocytes. The formation of clusters requires antigen-specific CD8(+) T cells and signaling by G protein-coupled receptors, although CD8(+) T cells of unrelated specificity are also recruited to clusters. By combining mathematical modeling and data analysis, we suggest that formation of clusters is mainly driven by enhanced recruitment of T cells into larger clusters. We further show various death phenotypes of the parasite, which typically follow prolonged interactions between infected hepatocytes and CD8(+) T cells. These findings stress the need for intravital imaging for dissecting the fine mechanisms of pathogen recognition and killing by CD8(+) T cells.

Induction and maintenance of protective CD8+ T cells against malaria liver stages: implications for vaccine development.

CD8+ T cells against malaria liver stages represent a major protective immune mechanism against infection. Following induction in the peripheral lymph nodes by dendritic cells (DCs), these CD8+ T cells migrate to the liver and eliminate parasite infected hepatocytes. The processing and presentation of sporozoite antigen requires TAP mediated transport of major histocompatibility complex class I epitopes to the endoplasmic reticulum. Importantly, in DCs this process is also dependent on endosome-mediated cross presentation while this mechanism is not required for epitope presentation on hepatocytes. Protective CD8+ T cell responses are strongly dependent on the presence of CD4+ T cells and the capacity of sporozoite antigen to persist for a prolonged period of time. While human trials with subunit vaccines capable of inducing antibodies and CD4+ T cell responses have yielded encouraging results, an effective anti-malaria vaccine will likely require vaccine constructs designed to induce protective CD8+ T cells against malaria liver stages.

Induction and maintenance of protective CD8+ T cells against malaria liver stages: implications for vaccine development

CD8+ T cells against malaria liver stages represent a major protective immune mechanism against infection. Following induction in the peripheral lymph nodes by dendritic cells (DCs), these CD8+ T cells migrate to the liver and eliminate parasite infected hepatocytes. The processing and presentation of sporozoite antigen requires TAP mediated transport of major histocompatibility complex class I epitopes to the endoplasmic reticulum. Importantly, in DCs this process is also dependent on endosome-mediated cross presentation while this mechanism is not required for epitope presentation on hepatocytes. Protective CD8+ T cell responses are strongly dependent on the presence of CD4+ T cells and the capacity of sporozoite antigen to persist for a prolonged period of time. While human trials with subunit vaccines capable of inducing antibodies and CD4+ T cell responses have yielded encouraging results, an effective anti-malaria vaccine will likely require vaccine constructs designed to induce protective CD8+ T cells against malaria liver stages.

Dendritic cells and hepatocytes use distinct pathways to process protective antigen from plasmodium in vivo.

Malaria-protective CD8+ T cells specific for the circumsporozoite (CS) protein are primed by dendritic cells (DCs) after sporozoite injection by infected mosquitoes. The primed cells then eliminate parasite liver stages after recognizing the CS epitopes presented by hepatocytes. To define the in vivo processing of CS by DCs and hepatocytes, we generated parasites carrying a mutant CS protein containing the H-2K(b) epitope SIINFEKL, and evaluated the T cell response using transgenic and mutant mice. We determined that in both DCs and hepatocytes CS epitopes must reach the cytosol and use the TAP transporters to access the ER. Furthermore, we used endosomal mutant (3d) and cytochrome c treated mice to address the role of cross-presentation in the priming and effector phases of the T cell response. We determined that in DCs, CS is cross-presented via endosomes while, conversely, in hepatocytes protein must be secreted directly into the cytosol. This suggests that the main targets of protective CD8+ T cells are parasite proteins exported to the hepatocyte cytosol. Surprisingly, however, secretion of the CS protein into hepatocytes was not dependent upon parasite-export (Pexel/VTS) motifs in this protein. Together, these results indicate that the presentation of epitopes to CD8+ T cells follows distinct pathways in DCs when the immune response is induced and in hepatocytes during the effector phase.

CD4+ T cells modulate expansion and survival but not functional properties of effector and memory CD8+ T cells induced by malaria sporozoites.

CD4(+) helper T cells are critical orchestrators of immune responses to infection and vaccination. During primary responses, naïve CD8(+) T cells may need "CD4 help" for optimal development of memory populations. The immunological factors attributed to CD4 help depend on the context of immunization and vary depending on the priming system. In response to immunization with radiation-attenuated Plasmodium yoelii sporozoites, CD8(+) T cells in BALB/c mice fail to generate large numbers of effector cells without help from CD4(+) T cells--a defect not observed in most systems. Given this unique early dependence on CD4 help, we evaluated the effects of CD4(+) cells on the development of functional properties of CD8(+) T cells and on their ability to abolish infection. First, we determined that this effect was not mediated by CD4(+) non-T cells and did not involve CD1d-restricted NKT cells. We found that CD8(+) T cells induced by sporozoites without CD4 help formed memory populations severely reduced in magnitude that could not limit parasite development in the liver. The inability of these "helpless" memory T cells to protect is not a result of defects in effector function, as their capacity to produce cytokines and undergo cytotoxic degranulation was indistinguishable from control memory T cells. These data indicate that CD4(+) T help may not be necessary to develop the functional attributes of CD8(+) T cells; however they are crucial to ensure the survival of effector and memory cells induced in primary responses.

Heterogeneous nuclear ribonucleoprotein E3 modestly activates splicing of tau exon 10 via its proximal downstream intron, a hotspot for frontotemporal dementia mutations.

The microtubule-associated protein tau is important to normal neuronal activity in the mammalian nervous system. Aggregated tau is the major component of neurofibrillary tangles (NFTs), structures present in the brains of people affected by neurodegenerative diseases called tauopathies. Tauopathies include Alzheimer's disease (AD), frontotemporal dementia with Parkinsonism (FTDP) and the early-onset dementia observed in Down syndrome (DS; trisomy 21). Splicing misregulation of adult-specific exon 10 results in expression of abnormal ratios of tau isoforms, leading to FTDP. Positions +3 to +19 of the intron downstream of exon 10 define a hotspot: Point mutations in it result in tauopathies. All these mutations increase exon 10 inclusion except for mutation +19, which almost entirely excludes exon 10. To investigate the tau connection between DS and AD, we examined splicing factors located on chromosome 21 for their effect on tau exon 10. By co-transfections, co-immunoprecipitations and RNAi constructs, we discovered that one of them, hnRNPE3 (PCBP3), modestly activates splicing of exon 10 by interacting with its proximal downstream intron around position +19. These results, coupled with the developmental profile of hnRNPE3, suggest a pathogenic role for splicing factors on chromosome 21 in neurodegenerative diseases with tangles and create a connection between tau splicing and the early-onset dementia of Down syndrome.

CpG-enhanced CD8+ T-cell responses to peptide immunization are severely inhibited by B cells.

Synthetic peptides encoding protective pathogen-derived epitopes represent--in principle--an ideal approach to T-cell vaccination. Empirically, however, these strategies have not been successful. In the current study, we profiled the early activation of CD8+ T cells by MHC class I-restricted peptide immunization to better understand the biology of this response. We found that CD8+ T cells proliferated robustly in response to low doses of short synthetic peptides in PBS, but failed to acquire effector function or form memory populations in the absence of the TLR ligand CpG. CpG was unique among TLR ligands in its ability to enhance the response to peptide and its adjuvant effects had strict temporal requirements. Interestingly, CpG treatment modulated T-cell expression of the surface receptors PD-1 and CD25, providing insight into its possible adjuvant mechanism. The effects of CpG on peptide immunization were dramatically enhanced in the absence of B cells, demonstrating a unique system of regulation of T-cell responses by these lymphocytes. The results reported here provide insight into the complex response to a simple vaccination regimen, as well as a framework for a rational peptide-based vaccine design to both exploit and overcome targeted aspects of the immune response.

Tau exon 6 is regulated by an intricate interplay of trans factors and cis elements, including multiple branch points.

Tau is a microtubule-associated protein whose transcript undergoes complex regulated splicing in the mammalian nervous system. Exon 6 of the gene is an alternatively spliced cassette whose expression profile differs from that of the other tau regulated exons, implying the involvement of distinct regulatory factors. Previous work had established the existence and use of two additional 3' splice sites within exon 6 and the influence of splicing factors polypyrimidine binding protein (PTB) and U2AF on its splicing. The present work shows that exon 6 isoforms exist in distinct ratios in different compartments of the nervous system and that splicing of exon 6 is governed by multiple branch points, exonic cis elements and additional trans factors. Recent results show that tau exon 6 is specifically suppressed in the brains of people who suffer from myotonic dystrophy type 1. The understanding of how tau exon 6 splicing is regulated may give us insights into the disease.

Identification, expression analysis, genomic organization and cellular location of a novel protein with a RhoGEF domain.

In this study we describe the identification and characterization of a novel cytosolic protein of the guanine exchange factor (GEF) family. The human cDNA corresponds to predicted human protein FLJ00128/FLJ10357 located on chromosome 14q11.2. The deduced protein sequence contains in its C-terminus a RhoGEF domain followed by a pleckstrin domain. Its N-terminus, central region and RhoGEF/pleckstrin domain are homologous to the recently identified zebrafish Quattro protein, which is involved in morphogenetic movements mediated by the actin cytoskeleton. Based on the homology of our protein's RhoGEF domain to the RhoGEF domains of Trio, Duo and Duet and its homology with Quattro, we named it Solo. The Solo mRNA is ubiquitously expressed but enriched in brain, its expression peaks perinatally and it undergoes extensive alternative splicing. In both myoblasts and neuroblastoma cells, the Solo protein is concentrated around the nucleus.

Saitohin, which is nested in the tau locus and confers allele-specific susceptibility to several neurodegenerative diseases, interacts with peroxiredoxin 6.

Saitohin is a gene unique to humans and their closest relatives, the function of which is not yet known. Saitohin contains a single polymorphism (Q7R), and its Q and R alleles belong to the H1 and H2 tau haplotype, respectively. The Saitohin Q allele confers susceptibility to several neurodegenerative diseases. To get a handle on Saitohin function, we used it as a bait in a yeast two-hybrid screen. By this assay and subsequent co-immunoprecipitation and glutathione S-transferase pull-down assays, we discovered and confirmed that Saitohin interacts with peroxiredoxin 6, a unique member of that family that is bifunctional and the levels of which increase in Pick disease. The strength of the interaction appeared to be allele-specific, giving the first distinction between the two forms of Saitohin.