Brian C Schaefer

Uniformed Services University of the Health Sciences | USUHS · Department of Microbiology & Immunology

Infections with rabies virus (RABV) and related lyssaviruses are uniformly fatal once virus accesses the central nervous system (CNS) and causes disease signs. Current immunotherapies are thus focused on the early, pre‐symptomatic stage of disease, with the goal of peripheral neutralization of virus to prevent CNS infection. Here, we evaluated the...

Rabies is a fatal zoonosis that is considered a re-emerging infectious disease. Although rabies remains endemic in canines throughout much of the world, vaccination programs have essentially eliminated dog rabies in the Americas and much of Europe. However, despite the goal of eliminating dog rabies in the European Union by 2020, sporadic cases of...

Rabies is a fatal zoonosis that is considered a re-emerging infectious disease. Although rabies remains endemic in canines throughout much of the world, vaccination programs have essentially eliminated dog rabies in the Americas and much of Europe. However, despite the goal of eradicating dog rabies in the European Union by 2020, sporadic cases of...

Bioluminescence imaging (BLI) is a technique that can be employed to quantify biological processes in living cells. When used in small animal models such as mice, BLI can provide both longitudinal and positional information regarding the biological process under investigation. Although perhaps best known for its utility in non-invasively quantifyin...

Rabies virus and related lyssaviruses cause uniformly fatal disease, once the infection progresses to the central nervous system. Current lyssavirus immunotherapies are directed toward peripheral neutralization of virus to prevent CNS infection during the pre-symptomatic stage of disease. In this study, using an anti-lyssavirus human monoclonal ant...

CARD19 is a mitochondrial protein of unknown function. While CARD19 was originally reported to regulate TCR-dependent NF-κB activation via interaction with BCL10, this function is not recapitulated ex vivo in primary murine CD8+ T cells. Here, we employ a combination of SIM, TEM, and confocal microscopy, along with proteinase K protection assays an...

Cell death plays a critical role in inflammatory responses. During pyroptosis, inflammatory caspases cleave Gasdermin D (GSDMD) to release an N-terminal fragment that generates plasma membrane pores that mediate cell lysis and IL-1 cytokine release. Terminal cell lysis and IL-1β release following caspase activation can be uncoupled in certain cell...

The adaptive immune system serves as a potent and highly specific defense mechanism against pathogen infection. One component of this system, the effector T cell, facilitates pathogen clearance upon detection of specific antigens by the T cell receptor (TCR). A critical process in effector T cell activation is transmission of signals from the TCR t...

Cell death plays a critical role in inflammatory responses. During pyroptosis, inflammatory caspases cleave Gasdermin D (GSDMD) to release an N-terminal fragment that generates plasma membrane pores that mediate cell lysis and IL-1 cytokine release. Terminal cell lysis and IL-1β release following caspase activation can be uncoupled in certain cell...

Australian bat lyssavirus (ABLV) is a rhabdovirus that circulates in four species of pteropid bats (ABLVp) and the yellow-bellied sheath-tailed bat (ABLVs) in mainland Australia. In the three confirmed human cases of ABLV, rabies illness preceded fatality. As with rabies virus (RABV), post-exposure prophylaxis (PEP) for potential ABLV infections co...

Australian bat lyssavirus (ABLV) is a rhabdovirus that circulates in four species of pteropid bats (ABLVp) and the yellow-bellied sheath-tailed bat (ABLVs) in mainland Australia. In the three confirmed human cases of ABLV, rabies illness preceded fatality. As with rabies virus (RABV), post-exposure prophylaxis (PEP) for potential ABLV infections co...

T cell responses to antigen are initiated by engagement of the T cell receptor (TCR)¹, leading to activation of diverse signaling cascades, including an incompletely defined pathway that triggers rapid remodeling of the actin cytoskeleton. Defects in control of actin dynamics and organization are associated with several human immunodeficiency disea...

After T cell receptor (TCR) engagement, the CARD11-Bcl10-Malt1 (CBM) complex oligomerizes to transduce NF-κB activating signals. Bcl10 is then degraded to limit NF-κB activation. The cDNA AK057716 (BinCARD-1) was reported to encode a novel CARD protein that interacts with Bcl10 and modestly inhibits NF-κB activation. In a later study, a second isof...

The adaptive immune system serves as a potent and highly specific defense mechanism against pathogen infection. One component of this system, the effector T cell, facilitates pathogen clearance upon detection of specific antigens by the T cell receptor (TCR). A critical process in effector T cell activation is transmission of signals from the TCR t...

Traditional mouse models of lyssavirus pathogenesis rely on euthanizing large groups of animals at various time points post-infection, processing infected tissues, and performing histological and molecular analyses to determine anatomical sites of infection. While powerful by some measures, this approach is limited by the inability to monitor disea...

CARD19 is a mitochondrial protein of unknown function; gene expression databases indicate that CARD19 is highly expressed in myeloid cells. We have observed that Card19 −/− mice injected with LPS display elevated levels of some pro-inflammatory cytokines relative to Card19 +/+ mice. Because CARD19 is a mitochondrial protein and mitochondrial dysfun...

The C-type lectin receptor–Syk (spleen tyrosine kinase) adaptor CARD9 facilitates protective antifungal immunity within the central nervous system (CNS), as human deficiency in CARD9 causes susceptibility to fungus-specific, CNS-targeted infection. CARD9 promotes the recruitment of neutrophils to the fungus-infected CNS, which mediates fungal clear...

Bats are increasingly implicated as hosts of highly pathogenic viruses. The underlying virus–host interactions and cellular mechanisms that promote co-existence remain ill-defined, but physiological traits such as flight and longevity are proposed to drive these adaptations. Autophagy is a cellular homeostatic process that regulates ageing, metabol...

The T cell receptor (TCR) to NF-κB signaling pathway is crucial for T cell activation and differentiation. Upon engagement of the TCR with cognate antigen, a series of events leads to the formation of the Carma1-Bcl10-Malt1 (CBM) complex, increasingly recognized as vital to a properly functioning immune response. We have demonstrated that in effect...

CARD19 is a ubiquitously expressed protein of unknown function. This protein was originally named BinCARD, and early data suggested that it interacts with Bcl10 and inhibits NF-κB activation in T cells. However, these early data were based on overexpression studies, and we are aware of no in vivo data that support this proposed function. Additional...

We have created an improved system for immortalizing mature macrophages by modifying the original v-raf/v-myc (J2) virus. Previously, these J2 viruses required a producer co-culture condition and maintained replicating virus. This provided more complexity, reagents, and biosafety risks. Our improved virus is replication deficient. Its lentiviral ge...

Overview of our T cell signaling research, suitable for readers with and without a science background.

Tumorigenesis is accompanied by broad changes to the surrounding tissue microenvironment in a tumor- and patient-specific manner. Phenotype shifts in tissue-resident immune cells promote cancer progression by establishing a proliferative inflammatory environment, activating angiogenesis, stimulating tumor cell invasion and suppressing anti-tumor im...

Tumorigenesis is modified by dynamic activities in the tumor microenvironment and mechanisms driving these alterations during tumor progression may be tumor- and patient-specific. Of well-established functions for tumor-associated stromal cells (e.g. angiogenesis, chemotaxis) immune system regulation by the tumor microenvironment is less well under...

T cell receptor (TCR) activation of the transcription factor NF-κB is a crucial determinant of effector T lymphocyte function. The complex regulatory network surrounding this pathway remains poorly understood, particularly at time periods further removed from initial TCR triggering. We have previously demonstrated that following activation of the T...

The T cell receptor (TCR) to NF-κB signaling pathway plays a critical role in regulation of proliferation and effector T cell differentiation and function. In naïve T cells, data suggest that most or all key cytoplasmic NF-κB signaling occurs in a TCR-proximal manner at the immunological synapse (IS). However, the subcellular organization of cytopl...

Neuroinflammation plays a critical role in the pathogenesis of traumatic brain injury (TBI). TBI induces rapid activation of astrocytes and microglia, infiltration of peripheral leukocytes, and secretion of inflammatory cytokines. In the context of modest or severe TBI, such inflammation contributes to tissue destruction and permanent brain damage....

Tight regulatory control of lymphocyte activation is necessary to avoid the deleterious consequences of an uncontrolled immune response. A complicated web of up-regulatory and down-regulatory processes governs a key activation pathway in lymphocytes, the antigen-receptor-to-NF-κB pathway; however, the complex interplay between positive and negative...

Using the parent-into-F1 model of induced lupus and (C57BL/6 × DBA2) F1 mice as hosts, we compared the inherent lupus-inducing properties of the two parental strain CD4 T cells. To control for donor CD4 recognition of alloantigen, we used H-2(d) identical DBA/2 and B10.D2 donor T cells. We demonstrate that these two normal, nonlupus-prone parental...

Human T lymphotropic virus type 1 (HTLV-1) trans-activator/oncoprotein, Tax, impacts a multitude of cellular processes, including I-κB kinase (IKK)/NF-κB signaling, DNA damage repair, and mitosis. These activities of Tax have been implicated in the development of adult T-cell leukemia (ATL) in HTLV-1-infected individuals, but the underlying mechani...

Human T lymphotropic virus type 1 (HTLV-1) trans-activator/oncoprotein, Tax, impacts a multitude of basic cellular processes, including I-κB kinase (IKK) signaling, DNA damage repair, and mitosis. These activities of Tax have been implicated in leukemogenesis, but the underlying mechanisms remain unknown. IKK and its upstream kinase, transforming g...

T cells are an immune cell lineage that play a central role in protection against pathogen infection. Antigen, in the form of pathogen-derived peptides, stimulates the T-cell receptor (TCR), leading to activation of the transcription factor, nuclear factor kappa B (NF-κB). The subsequent NF-κB-dependent gene expression program drives expansion and...

Antigen-mediated stimulation of the T cell receptor (TCR) triggers activation of nuclear factor κB (NF-κB), a key transcriptional regulator of T cell proliferation and effector cell differentiation. TCR signaling to NF-κB requires both the Carma1-Bcl10-Malt1 (CBM) complex and the inhibitor of κB (IκB) kinase (IKK) complex; however, the molecular me...

Antigen stimulation of T cell receptor (TCR) signaling to nuclear factor (NF)-κB is required for T cell proliferation and differentiation of effector cells. The TCR-to-NF-κB pathway is generally viewed as a linear sequence of events in which TCR engagement triggers a cytoplasmic cascade of protein-protein interactions and post-translational modific...

T cells are essential for defense of the host against invading pathogens. Antigen activation of the T cell receptor (TCR) is required for generation of an adaptive immune response. Several groups have observed that blocking autophagy augments T cell activation, but the molecular basis of this finding has remained elusive. The adaptor protein BCL10...

An immediate consequence of traumatic brain injury (TBI) is the induction of an inflammatory response. Mounting data suggest that inflammation is a major contributor to TBI-induced brain damage. However, much remains unknown regarding the induction and regulation of the inflammatory response to TBI. In this study we compared the TBI-induced inflamm...

The adaptor protein Bcl10 is a critically important mediator of T cell receptor (TCR)-to-NF-κB signaling. Bcl10 degradation is a poorly understood biological phenomenon suggested to reduce TCR activation of NF-κB. Here we have shown that TCR engagement triggers the degradation of Bcl10 in primary effector T cells but not in naive T cells. TCR engag...

The adaptor protein Bcl10 is a critically important mediator of T cell receptor (TCR)-to-NF-κB signaling. Bcl10 can be degraded by TCR-dependent proteolysis, but the molecular mechanism of degradation is poorly understood. We show that TCR engagement triggers the degradation of Bcl10 in primary effector T cells, but not in naïve T cells. TCR engage...

Protein kinase Cθ (PKCθ) is a serine/threonine kinase that plays an essential role in antigen-regulated responses of T lymphocytes. Upon antigen stimulation, PKCθ is rapidly recruited to the immunological synapse (IS), the region of contact between the T cell and antigen-presenting cell. This behavior is unique among T cell PKC isoforms. To define...

TCR-mediated activation of the transcription factor NF-κB is required for T cell proliferation, survival, and effector differentiation. Although this pathway is the subject of intense study, it is not known whether TCR signaling to NF-κB is digital (switch-like) or analog in nature. Through analysis of the phosphorylation and degradation of IκBα an...

Differential gene expression in liver tissue of RAG-1-/- and OT-II/RAG-1-/- mice. (0.29 MB DOC)

Biological Functions associated with genes that are differentially expressed in liver tissue of RAG-1-/- and OT-II/RAG-1-/- mice. (0.35 MB DOC)

CD4+ T cell activation in response to S. mansoni infection is impaired in Bcl10-/- mice. (A) Activation marker expression by hepatic or (B) splenic CD4+ T cells was evaluated in response to pre-patent S. mansoni infection 28 days post-infection. Surface expression of CD44 and CD62L, CD69, and CD25 on gated CD4+TCRβ+NK1.1- cells is shown. Numbers re...

CD4+ T cell activation in response to S. mansoni antigens is impaired in OT-II/RAG-1-/- mice. (A) Activation marker expression by hepatic and splenic CD4+ T cells in wild type and OT-II/RAG-1-/- mice was evaluated in response to pre-patent S. mansoni infection 28 days post-infection. Surface expression of CD44 and CD62L on gated CD4+TCRβ+NK1.1- cel...

Schistosoma blood flukes, which infect over 200 million people globally, co-opt CD4+ T cell-dependent mechanisms to facilitate parasite development and egg excretion. The latter requires Th2 responses, while the mechanism underpinning the former has remained obscure. Using mice that are either defective in T cell receptor (TCR) signaling or that la...

Malt1 is a multi-domain cytosolic signaling molecule that was originally identified as the target of recurrent translocations in a large fraction of MALT lymphomas. The product of this translocation is a chimeric protein in which the N-terminus is contributed by the apoptosis inhibitor, cIAP2, and the C-terminus is contributed by Malt1. Early studi...

Confocal microscopy is a widely employed methodology in cellular biology, commonly used for investigating biological organization at the cellular and sub-cellular level. Most basic confocal microscopes are equipped to cleanly discriminate no more than four fluorophores in a given sample, limiting the utility of this method for co-localization, co-e...

The cytosolic proteins protein kinase Ctheta (PKCtheta), Bcl10, and Malt1 play critical roles in TCR signaling to the transcription factor NF-kappaB. Our data confirm that CD4(+) T cells from PKCtheta, Bcl10, and Malt1 knockout mice show severe impairment of proliferation in response to TCR stimulation. Unexpectedly, we find that knockout CD8(+) T...

The cytosolic proteins protein kinase CO (PKC theta), Bcl10, and Malt1 play critical roles in TCR signaling to the transcription factor NF-kappa B Our data confirm that CD4(+) T cells from PKC theta, Bcl10, and Malt1 knockout mice show severe impairment of proliferation in response to TCR stimulation. Unexpectedly, we find that knockout CD8(+) T ce...

Bcl10 and MALT1 are essential mediators of NF-kappaB activation in response to the triggering of a diverse array of transmembrane receptors, including antigen receptors. Additionally, both proteins are translocation targets in MALT lymphoma. Thus, a detailed understanding of the interaction between these mediators is of considerable biological impo...

The Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) is a functional homologue of the tumor necrosis factor receptor family and contributes substantially to the oncogenic potential of EBV through activation of nuclear factor kappaB (NF-kappaB). MicroRNAs (miRNAs) are a class of small RNA molecules that are involved in the regulatio...

Stimulation of the T-cell receptor (TCR) results in the activation of several transcription factors, including NF-kappaB, that are crucial for T-cell proliferation and gain of effector functions. On TCR engagement, several proteins within the TCR-directed NF-kappaB signaling pathway undergo dynamic spatial redistribution, but the significance of th...

The green fluorescence protein (GFP) from the UBI-GFP/BL6 transgenic line was bred into C57BL/6J-scid and C.B-17-scid mice for investigating host-tumor cell interactions. These mice express high levels of GFP under the control of the ubiquitin promoter in virtually all cells examined. In tumor tissue generated by implanting tumor cells in the GFP t...

Development of many vertebrate tissues involves long-range cell migrations. In most cases, these migrations have been inferred from analysis of single time points and the migration process has not been directly observed and quantitated in real time. In the mammalian adult thymus, immature CD4+ CD8+ double-positive (DP) thymocytes are found in the o...

Orientation of Imaging Relative to Thymic Lobes In Vivo Thymic lobes are depicted in their normal position relative to the heart. Thymic lobes were surgically removed and separated, and then the dorsal side (side facing the heart) of thymic lobe was adhered to glass cover slip. Imaging (see Figure S3A and S3B) was performed with the objective posit...

GFP Thymocytes within an Intact Thymic Lobe A representative 3D image of GFP thymocytes within an intact thymic lobe. Image is rendered from one z stack at a single time point and is shown in a 360° rotation. Image size is 164 × 164 × 40 μm. Image was recorded approximately 140 μm below the thymic capsule. Corresponds to Figure 1. (3.9 MB ZIP).

Frequency Distribution of Average Motility Rates for Wild-Type Cortical Thymocytes Histograms showing the frequency distribution of average motility rates for wild-type cortical thymocytes were obtained from four individual runs. Compiled data are shown in Figure 2A. (76 KB TIF).

Displacement Analyses of Wild-Type MRhi Cells Results of displacement analyses of wild-type MRhi cells from 4 individual experiments are shown. Bar graphs show the average displacement per MRhi cell moved in each direction in a 3-min interval. Data shown were computed from 11–16 MRhi cells from each dataset. The four runs made up two separate stack...

Frequency Distribution of Average Motility Rates for P14 Cortical Thymocytes Histograms showing the frequency distribution of average motility rates for P14 cortical thymocytes were obtained from four individual runs. Compiled data are shown in Figure 4A. (79 KB TIF).

4D Tracking of Wild-Type GFP Thymocytes Migrating through an Intact Thymic Lobe Same dataset as shown in Video S2 with tracks highlighted. Tracks were generated using 4D cell-tracking software. The fluorescence signal from GFP thymocytes is shown in green, and the positions of individual cells as determined by tracking software are represented as g...

P14 TCR Transgenic GFP Thymocytes in an Intact Thymic Lobe Time-lapse image of P14 TCR transgenic GFP thymocytes in an intact thymic lobe. The P14 TCR induces positive selection in this system. Note that a high proportion of thymocytes migrate rapidly and in straight trajectories compared to wild-type GFP thymocytes (Videos S2 and S3). Corresponds...

P14 GFP Thymocyte Migration is Biased in the z Direction Time-lapse image of P14 TCR transgenic GFP thymocytes in intact thymic lobe is shown rotated to display the x and z dimensions. The same dataset was used to generate Video S6. Tracks of MRhi cells are highlighted. Note that the majority of MRhi tracks are oriented in the z direction. (1.3 MB...

Developmental Profiles of GFP+ Thymocytes from Chimeric Mice Representative profiles obtained by flow cytometric analysis of P14 and 5CC7 chimeric thymii. As expected, GFP+-gated P14 thymocytes (top row) showed high levels of TCR within the CD4+CD8+ population and a high percentage of CD8+ SP thymocytes, indicating a high frequency of positive sele...

Two-Photon Imaging of Thymocyte Migration in Intact Thymic Lobes (A) Explanted GFP chimeric thymic lobe was placed in oxygen-perfused media and maintained at 37 °C throughout experiment. Objective was placed directly over the top of lobe and a total of 20 optical slices at 2-μm step intervals were acquired, which generated z stacks of 104 x 104 x 4...

Displacement Analyses of P14 MRhi Cells Results of displacement analyses of P14 MRhi cells from four individual experiments are shown. Bar graphs show the average displacement per MRhi cell in each direction in a 3-min interval. Data shown were computed from 29–35 MRhi cells from each dataset. Compiled data are shown in Figure 4C. (76 KB TIF).

Wild-Type GFP Thymocytes Migrating through an Intact Thymic Lobe Time-lapse image of dataset used to generate Video S1. Image is shown as a maximum projection of all z stacks. Corresponds to Figure 1. All movies were generated from 20 to 33 min of imaging and are played at six frames per second unless otherwise indicated. (2 MB ZIP).

MRhi Cell Propulsion Is Associated with Polarized Morphology Time-lapse image of GFP thymocytes cropped to approximately 40 × 40 × 40 μm in the x, y, and z directions. Note the polarized morphology and dramatic shape changes of the MRhi cell as it crawls from bottom to upper left corner. Video shown was generated from 5 min of imaging and is played...