Biology subjects

Pfaller, C. K.

Publications and source records attributed to Pfaller, C. K..

8 recordsLinked to original sources

bioRxiv2025

Antigen-specific CD8 T cells are generated and reactivated in the bone marrow following viral brain infections and impact the bone marrow niche

Bone marrow is the primary immune organ responsible for stem cell maintenance and hematopoiesis. However, the contribution of the bone marrow niche for the generation of adaptive immune responses is less well-understood. We therefore assessed the capacity of virus antigen-specific CD8 T cells to be generated and expanded in bone marrow following acute neurotropic virus infection. Intracranial infection with the neurotropic Theilers Murine Encephalomyelitis Virus (TMEV) results in an acute infection in the C57BL/6 mouse which is cleared within 4 weeks post infection due to the generation of an antigen-specific CD8 T cell response against the immunodominant epitope VP2121-130. We determined that 1-10% of CD8 T cells present in the femoral, and sternal bone marrow were virus antigen-specific 5-7 days after intracranial TMEV infection. We determined that antigen-specific CD8 T cells were generated in the bone marrow with similar kinetics to that of conventional responses in the secondary lymphoid organs. Importantly, continuous treatment with FTY720, which sequesters T cells outside of the blood, did not eliminate antigen-specific CD8 T cells in the bone marrow compartment indicating generation within the bone marrow niche. Similarly, injection of Poly I:C admixed with fluorescently labeled ovalbumin into the brain generated a similar antigen-specific CD8 T cell response in the bone marrow indicating these responses occur following various brain insults. This model also identified the likely antigen presenting cell (APC) responsible for scavenging antigens from the brain and trafficking into the bone marrow as a migratory myeloid-derived APC. Moreover, following clearance of TMEV infection, CD8 T cells in the bone marrow established durable memory. Antigen-specific memory CD8 T cells in the bone marrow reactivated and expanded upon cognate antigen reencounter. Antigen-specific reactivation of memory CD8 T cells within the bone marrow caused a concurrent increase in Lineage-, Sca-1+, c-Kit+ (LSK) and CD11b+ MHCII+ myeloid cells. This increase in the LSKs and myeloid cells in the bone marrow niche was abrogated with CD8 T cell depletion. We conclude that brain viral infections induce in situ effector and memory T cell responses within the bone marrow compartment. Memory recall CD8 T cell responses induce niche dysregulation by expanding LSK cells and lead to an influx of MHCII+ myeloid cells. Our data pave the way for crucial studies of bone marrow resident antigen-specific CD8 T cells in health and diseases.

immunology↗

bioRxiv2025

Measles vaccine attenuation comprises innate immune cell infection and interferon responses

Measles virus (MeV) is a highly contagious pathogen capable of infecting immune cells and thereby causing immunosuppression and immune amnesia. A live-attenuated MeV vaccine is highly efficacious and has been safely administered for over 60 years. However, a detailed mechanistic understanding of the mode of its attenuation remains elusive. Here, we determined the molecular mechanisms underlying MeV vaccine attenuation in primary human immune cells. Enhanced infection of innate immune cells resulting in uncontrolled viral RNA synthesis and reduced ability to evade host antiviral responses induced a strong type-I interferon signature that arrested replication of the vaccine strain. In contrast, the wild type virus replicated slowly but steadily, avoiding detection by innate immunity.

immunology↗

bioRxiv2024

TSniffer: Unbiased de novo identification of RNA editing sites and quantification of editing activity in RNA-seq data

RNA editing by adenosine deaminases acting on RNA (ADARs) is an evolutionarily conserved posttranscriptional modification essential for organismal development and normal cell function. Three catalytically active ADARs are conserved in mammals: two isoforms of ADAR1 referred to as ADAR1-p150 and ADAR1-p110, as well as ADAR2. All recognize and edit double-stranded RNA (dsRNA) structures but demonstrate target specificity and selectivity that dictate the unique essential biological functions of the three enzymes. The editing activity of ADAR1-p150 suppresses autoimmune responses against self-RNA structures, whereas ADAR1-p110 and ADAR2 have other primary functions. To better understand the mechanism of target selection by ADARs, we developed TSniffer, which allows accurate de novo identification of edited transcripts and quantification of the extent of editing within each transcript. We found that 17-40% of protein coding transcripts in mice, ferrets, and humans are edited by ADARs. Individual transcripts can harbor hundreds and thousands of editing sites, mostly within inverted retrotransposable elements. For human transcripts, we found differential editing by ADAR1 and ADAR2, aligning with a supportive role for ADAR2, while some targets were dominantly edited by ADAR1. Relying only on RNA-seq data and reference genome, TSniffer represents a novel tool to decipher the role of ADAR editing in different physiological states including disease models. Its unbiased approach is suitable for any organism.

bioinformatics↗

bioRxiv2022

Non-neutralizing antibody responses to vesicular stomatitis virus-vectored influenza A virus vaccines correlate with protection

Seasonal influenza virus infections remain a major global public health burden. In addition, influenza A virus (IAV) exhibits high pandemic potential through zoonotic spread from avian hosts to humans. Currently licensed IAV vaccines are mainly directed against the immuno-dominant surface protein hemagglutinin (HA). Since HA is antigenically highly variable, IAV can escape vaccine-derived immunity through antigenic drift. However, vaccine preparations such as the live-attenuated influenza vaccine (LAIV) also contain other more conserved viral antigens, whose contribution to influenza immunity are not fully elucidated. To determine the extent to which conserved LAIV antigens contribute to establishing protective immunity against heterologous IAV strains, we generated vesicular stomatitis virus-based single-round vector vaccines expressing individual LAIV antigens, and tested their ability to protect mice from a heterologous challenge with two IAV strains, PR8[H1N1] and rSC35M[H7N7]. We found that immunization with nucleoprotein (NP), ion channel M2, and the stem-region of HA (HAstem), but not matrix protein (M1), provide protection from severe disease caused by either IAV strain. This effect correlated with development of non-neutralizing antibodies cross-reactive with both virus strains. Notably, the individual antigens induced specific IgG subclass profiles with different reactivity against PR8 and rSC35M. Sera from vaccinated animals activated Fc-gamma receptor IV-mediated effector functions, suggesting that they can induce cell-mediated immune defense mechanisms, such as antibody-dependent cellular cytotoxicity and antibody-dependent cellular phagocytosis. Combination of the most potent antigens NP and M2 in a mixed vaccination resulted in enhanced protection against IAV challenge, suggesting that the antibody responses against these antigens were synergistic. Our results demonstrate the potency of NP and M2 proteins to serve as conserved antigen targets, resulting in broad protection against severe IAV disease.

immunology↗

bioRxiv2022

Brain resident memory T cells rapidly expand and initiate neuroinflammatory responses following CNS injury and viral infection

The contribution of circulating verses tissue resident memory T cells (TRM) to clinical neuropathology is an enduring question due to a lack of mechanistic insights. The prevailing view is TRM cells are protective against pathogens in the brain. However, the extent antigen-specific TRM cells can induce neuropathology upon reactivation has not been determined. Using the described phenotype of TRMs, we found that brains of naive mice harbor populations of CD69+ CD103- T cells. Notably, numbers of CD69+ CD103- TRM cells rapidly increase following neurological insults of physical, cancerous, or viral origins. This TRM expansion precedes infiltration of virus specific CD8 T cells and is due to proliferation of T cells within the brain. In contrast, the CD69+ CD103+ TRMs in the brain are generated after the initial expansion of CD69+ CD103- cells following injury and are antigen-specific. We next evaluated the capacity of antigen-specific TRMs in the brain to induce significant neuroinflammation post virus clearance, including infiltration of inflammatory monocytes, activation of T cells in the brain, and significant blood brain barrier disruption. These neuroinflammatory events were induced by TRMs, as depletion of peripheral T cells or blocking T cell trafficking using FTY720 did not change the neuroinflammatory course. Reactivation of antigen-specific TRMs in the brain also induced profound lymphopenia within the blood compartment. We have therefore determined that antigen-specific TRMs can induce significant neuroinflammation, neuropathology, and peripheral immune suppression. Importantly, understanding functions of brain TRMs is crucial in investigating their role in neurodegenerative disorders, CNS cancers, and long-term complications associated with viral infections including COVID-19. Graphical AbstractHealthy brain harbors populations of resident memory T cells (TRM). These TRM cells rapidly proliferate in response to CNS insults of various origins. Following clearance of the insult, populations of TRM cells in the brain decline, but an antigen-specific TRM subset remains within the brain. Antigen-specific reactivation of brain TRMs mediates neuroinflammatory sequalae involving activation and blasting of resident T cells, infiltration of inflammatory monocytes and blood brain barrier disruption. Severe neuroinflammation within the brain following antigen-specific TRM reactivation is concurrent with profound lymphopenia within the blood compartment. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=132 SRC="FIGDIR/small/487707v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@a22867org.highwire.dtl.DTLVardef@a90e0org.highwire.dtl.DTLVardef@1ad904eorg.highwire.dtl.DTLVardef@1dd281c_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2021

Initial HCV infection of adult hepatocytes triggers a temporally structured transcriptional program containing diverse pro- and anti-viral elements

Transcriptional profiling provides global snapshots of virus-mediated cellular reprogramming, which can simultaneously encompass pro- and antiviral components. To determine early transcriptional signatures associated with HCV infection of authentic target cells, we performed ex vivo infections of adult primary human hepatocytes (PHHs) from seven donors. Longitudinal sampling identified minimal gene dysregulation at six hours post infection (hpi). In contrast, at 72 hpi, massive increases in the breadth and magnitude of HCV-induced gene dysregulation were apparent, affecting gene classes associated with diverse biological processes. Comparison with HCV-induced transcriptional dysregulation in Huh-7.5 cells identified limited overlap between the two systems. Of note, in PHHs, HCV infection initiated broad upregulation of canonical interferon (IFN)-mediated defense programs, limiting viral RNA replication and abrogating virion release. We further find that constitutive expression of IRF1 in PHHs maintains a steady-state antiviral program in the absence of infection, which can additionally reduce HCV RNA translation and replication. We also detected infection-induced downregulation of [~]90 genes encoding components of the EIF2 translation initiation complex and ribosomal subunits in PHHs, consistent with a signature of translational shutoff. As HCV polyprotein translation occurs independently of the EIF2 complex, this process is likely pro-viral: only translation initiation of host transcripts is arrested. The combination of antiviral intrinsic and inducible immunity, balanced against pro-viral programs, including translational arrest, maintains HCV replication at a low-level in PHHs. This may ultimately keep HCV under the radar of extra-hepatocyte immune surveillance while initial infection is established, promoting tolerance, preventing clearance and facilitating progression to chronicity. IMPORTANCEAcute HCV infections are often asymptomatic and therefore frequently undiagnosed. We endeavored to recreate this understudied phase of HCV infection using explanted PHHs and monitored host responses to initial infection. We detected temporally distinct virus-induced perturbations in the transcriptional landscape, which were initially narrow but massively amplified in breadth and magnitude over time. At 72 hpi, we detected dysregulation of diverse gene programs, concurrently promoting both virus clearance and virus persistence. On the one hand, baseline expression of IRF1 combined with infection-induced upregulation of IFN-mediated effector genes suppresses virus propagation. On the other, we detect transcriptional signatures of host translational inhibition, which likely reduces processing of IFN-regulated gene transcripts and facilitates virus survival. Together, our data provide important insights into constitutive and virus-induced transcriptional programs in PHHs, and identifies simultaneous antagonistic dysregulation of pro-and anti-viral programs which may facilitate host tolerance and promote viral persistence.

immunology↗

bioRxiv2020

Safe and effective two-in-one replicon-and-VLP minispike vaccine for COVID-19

The large SARS-CoV-2 spike (S) protein is the main target of current COVID-19 vaccine candidates but can induce non-neutralizing antibodies, which may cause vaccination-induced complications or enhancement of COVID-19 disease. Besides, encoding of a functional S in replication-competent virus vector vaccines may result in the emergence of viruses with altered or expanded tropism. Here, we have developed a safe single round rhabdovirus replicon vaccine platform for enhanced presentation of the S receptor-binding domain (RBD). Structure-guided design was employed to build a chimeric minispike comprising the globular RBD linked to a transmembrane stem-anchor sequence derived from rabies virus (RABV) glycoprotein (G). Vesicular stomatitis virus (VSV) and RABV replicons encoding the minispike not only allowed expression of the antigen at the cell surface but also incorporation into the envelope of secreted non-infectious particles, thus combining classic vector-driven antigen expression and particulate virus-like particle (VLP) presentation. A single dose of a prototype replicon vaccine, VSV{Delta}G-minispike-eGFP (G), stimulated high titers of SARS-CoV-2 neutralizing antibodies in mice, equivalent to those found in COVID-19 patients. Boost immunization with the identical replicon further enhanced neutralizing activity. These results demonstrate that rhabdovirus minispike replicons represent effective and safe alternatives to vaccination approaches using replication-competent viruses and/or the entire S antigen. HighlightsO_LISARS-CoV-2 S RBD antigen is preferred over entire S to preclude potential disease enhancing antibodies C_LIO_LIconstruction of a chimeric rhabdovirus minispike protein presenting RBD in native conformation C_LIO_LIconstruction of single round VSV and rabies virus replicon vaccines C_LIO_LIpresentation of minispike antigen on cells and on noninfectious VLPs C_LIO_LIstrong induction of SARS-CoV-2 neutralizing antibodies by the VSV replicon/VLP system in vaccinated mice C_LI

microbiology↗

bioRxiv2020

Severe and multifaceted systemic immunosuppression caused by experimental cancers of the central nervous system requires release of non-steroid soluble mediators

Immunosuppression of unknown etiology is a hallmark feature of glioblastoma (GBM) and is characterized by decreased CD4 T cell counts and down regulation of MHC class II expression on peripheral blood monocytes in patients. This immunosuppression is a critical barrier to the successful development of immunotherapies for GBM. We recapitulated the immunosuppression observed in GBM patients in the C57BL/6 mouse and investigated the etiology of low CD4 T cell counts. We determined that thymic involution was a hallmark feature of immunosuppression in three distinct models of CNS cancer, including mice harboring GL261 glioma, B16 melanoma, and in a spontaneous model of Diffuse Intrinsic Pontine Glioma (DIPG). In addition to thymic involution, we determined that tumor growth in the brain induced significant splenic involution, reductions in peripheral T cells, reduced MHC class II expression on hematopoietic cells, and a modest increase in bone marrow resident CD4 T cells with a naive phenotype. Using parabiosis we report that thymic involution, declines in peripheral T cell counts, and reduced MHC class II expression levels were mediated through circulating blood-derived factors. Conversely, T cell sequestration in the bone marrow was not governed through circulating factors. Serum isolated from glioma-bearing mice potently inhibited proliferation and functions of T cells both in vitro and in vivo. Interestingly, the factor responsible for immunosuppression in serum is nonsteroidal and of high molecular weight. Through further analysis of neurological disease models, we determined that the aforementioned immunosuppression was not unique to cancer itself, but rather occurs in response to CNS injury. Noncancerous acute neurological insults also induced significant thymic involution and rendered serum immunosuppressive. Both thymic involution and serum-derived immunosuppression were reversible upon clearance of brain insults. These findings demonstrate that CNS cancers cause multifaceted immunosuppression and pinpoint circulating factors as a target of intervention to restore immunity. Short SummaryCNS cancers and other brain-injuries suppress immunity through release of non-steroid soluble factors that disrupt immune homeostasis and dampen responses of the peripheral immune system. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/006825v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@14e90c2org.highwire.dtl.DTLVardef@101de5corg.highwire.dtl.DTLVardef@1ad8534org.highwire.dtl.DTLVardef@1041fef_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

immunology↗