bluuebunny/crossref_metadata_2025 huggingface dataset:
Ubiquitin Signaling: Systematic Profiling in the Immune Response
Authors: Song Changcheng , Lu Xiaolong , Akimov Vyacheslav , Singh Rajesh , Mason Mark , Blagoev Blagoy
Published: January 2023

Cell signaling induced by cytokine receptors is regulated by ubiquitin (Ub) modification. Ub forms polymerized chains to encrypt the coding for the alteration of signaling molecules in immune response. Here we report a systematic approach for profiling Ub coding. Ubiquitylation of specific target proteins in stimulated and unstimulated human PBMCs and T Leukemia (Jurkat) cells are profiled by ELISA and Immunoblotting UBiTest and Ubi-Omics methods. The cells are treated with the de-ubiquitylase USP7 inhibitor P50378, proteasome inhibitor bortezomib, or a ubiquitin ligase inhibitor. Poly-ubiquitylated cellular proteins is affinity isolated using Tetra Ub Binding Entities (TUBEs), which bind Ub with high affinity (nmol). TUBEs that are selective for isolated K48, K63 and M1 (linear) poly-ubiquitylated proteins are employed. Then, these polyubiquitylated proteins were digested with Lys-C and immunoprecipitated with UbiSite antibody. Ub-rich peptides were analyzed by LC-MS/MS. This UbiSite-Omics approach affords the advantages of both identifying N-terminal ubiquitylation and distinguishing ubiquitination from modification by Ub-like proteins. These data provide proteome-wide profiling of Ub-omics not achievable by traditional proteomics methods and suggest that the TUBE and UbiSite based technology will reveal new Ub landscapes in immune cells. This novel approach will facilitate the discovery of pharmacological markers for drugs, and enable cell biologists to identify patterns of polyUb proteins.
Effects of temporary BLyS treatment of rhesus macaques
Authors: Scholz Jean , Pahar Bapi , Madej Joanna B , Soldemo Martina , Trunch Wendy Lala , Guenaga Javier , Myles Arpita , Tran Karen , Wyatt Richard T , Hedestam Gunilla B Karlsson , Lackner Andrew , Cancro Michael
Published: January 2023

BLyS is a survival cytokine that regulates peripheral B cell numbers and transitional B cell throughput. In mice, exogenous BLyS administration yields elevated pre-immune B cell numbers, an increased proportional representation of transitional B cells, and shifts in repertoire composition (1–3). Consistent with these observations, BLyS pretreatment alters the quality of antibody responses to HIV gp140 in mice, yielding enhanced neutralizing responses (4). While it is assumed that BLyS plays analogous roles in other species, similar studies in nonhuman primates are lacking. Accordingly, we have assessed the effects of exogenous BLyS treatment in rhesus macaques. Here we treated juvenile rhesus macaques with 0.05 mg/kg recombinant human BLyS over ten days. Physical exams and blood cell counts indicate no adverse effects of treatment except a decrease in lymphocyte counts during the treatment periods. We find that the numbers and proportional representation of transitional B cells (CD20+ IgM+ CD10+) increase during and immediately following BLyS treatment, returning to pre-treatment levels within 20–30 days. There is a small increase in plasma anti-dsDNA antibody, and a significant increase in anti-recombinant human BLyS antibody indicating immunogenicity in this species. Together, these results indicate that BLyS plays similar roles in rodents and primates. They further suggest that deliberate manipulation of BLyS may be an effective approach in rhesus macaques for expanding B cell repertoire diversity and altering the quality of antibody responses.
Estimate of within clones and between clone selection suggests a multi-stage selection process in the production of high affinity B cell receptors.
Authors: Benichou Jennifer Israel Cohen , Louzoun Yoram
Published: January 2023

During an immune response, B cells undergo a process of clonal expansion, somatic hypermutation of the immunoglobulin genes and affinity-dependent selection. Over a lifetime, each B cell may participate in multiple rounds of affinity maturation as part of different immune responses. We characterized these two time-scales for selection which are apparent in the structure of B cell lineage trees. The “trunk” of the tree consists of mutations that are shared across all members of a clone, and the branches form a “canopy” consisting of mutations that are shared by a subset of clone members.

We show that the framework regions are subjected to a negative selection at all stages. However, CDR regions are affected to purifying and antigen-driven positive selection on the short term, which leads to a net positive selection in the long term. Overall, we found that long-term selection is strongly dependent on the heavy chain variable gene family.

To the selection in the tree canopy, we propose a novel mixed lineage tree/sequence based method to detect within population selection as defined by the effect of mutations on the average number of offspring. Specifically, we propose to measure the log of the ratio between the number of leaves in lineage trees branches following synonymous and non-synonymous mutations (LONR), and show that selection in CDR region occur at a different stage.
Adenosine regulation of danger signaling
Authors: Cekic Caglar , Akdemir Imran , Koyas Altay , Kayhan Merve , Savas Ali Can
Published: January 2023

Metabolic and immune related activities converge as main triggers of adenosine accumulation in extracellular space. Adenosine by engaging adenosine A2A and A2B receptors strongly suppresses innate and adaptive immune responses. Although adenosine receptors are being targeted in preclinical and clinical studies how different danger signals are regulated by adenosine is poorly understood. Here we showed that adenosine receptor stimulation strongly inhibited inflammatory responses while sparing type-I interferon responses downstream of different danger signals in dendritic cells and macrophages. Mechanistically, danger signals associated with MyD88-dependent inflammatory pathways such as LPS and CpG but not the danger signals associated with IRF3/Type-I interferon pathways such as pA:U and cGAMP increase the expression adenosine A2A and A2B receptors. Adenosine was shown to increase the expression of NR4A nuclear hormone receptors to inhibit NF-κB activation. Although adenosine did not influence NF-κB phosphoactivation expression of anti-inflammatory NR4A1 was increased after adenosine receptor stimulation in the presence of TLR ligands known to activate MyD88 pathway but not in the presence of cGAMP and pA:U. Overall these results indicate that there is a differential modulation of danger signaling by adenosine rather than overall supression. Our results have important implications for developing combinatorial approaches to target adenosine and danger signaling pathways to cure immune-related diseases.
The role of SHP-2 on NK cell development and functions
Authors: Miah S.M. Shahjahan , Sexl Veronica , Yang Wentian , Brossay Laurent
Published: January 2023

The SH2 domain-containing tyrosine phosphatase SHP-2 (PTPN11) has been shown to play essential roles in immune cell signaling. Several inhibitory receptors expressed on natural killer (NK) cells associate with SHP-2 to exert downstream functions. Here we examined the influence of SHP-2 on NK cell development and effector activity. Because SHP-2 is required for embryonic development in mice, we generated mice conditionally deficient for SHP-2 in NK cells. SHP-2fl/fl mice were crossed to NKp46-Cre mice in order to inactivate Ptpn11 in NKp46+ cells. These mice were also crossed to R26R-EYFP reporter mice to track cells with active Cre recombinase. We found an increase in NK cell frequency and number in these mice compared to littermate controls. The frequency of mature NK cells (CD11b+CD27−) was also increased in the periphery while decreased in the bone marrow. In addition, the homeostatic proliferation of SHP-2-deficient NK cells was impaired. However, effector functions were not affected significantly. These included IFN-γ production at the peak of the NK cell response during MCMV infection, rejection of β2-microglobulin deficient splenocytes, and degranulation via Ly49H using M157-transfected cells as targets. Taken together, our data suggest that SHP-2 is dispensable for NK cell effector functions and education, but is important for NK cell maturation.
Reactive oxygen species modulate immune cell effector function
Authors: Lee Seung-Hwan , Almutairi Saeedah , Ali Alaa Kassim
Published: January 2023

During an infection or carcinogenesis, the stimulation of immune cells may lead to elevated intracellular levels of reactive oxygen species (ROS), and this oxidative stress can cause damage to the cell. Interestingly, previous reports have shown that ROS produced through cell activation play a critical role as signaling messengers in immune responses. Yet, little is known in regard to the role of ROS in natural killer (NK) cell effector functions. Our data suggest that the levels of ROS increase in NK cells upon stimulation. Therefore, we hypothesize that, upon stimulation of NK cells, ROS accumulate and provide positive signaling that leads to the upregulation of NK cell effector functions. To test this hypothesis, we examined NK cell function and proliferation in Glrx2-deficient mice in which the ROS levels are elevated in the mitochondrial matrix due to a defect in the redox system. We found that lack of Glrx2 in NK and T cells does not improve their cytotoxic potential or their ability to produce IFNg. However, Glrx2-deficient NK cells showed enhanced proliferation when stimulated with IL-2, indicating that during prolonged stress, the elevated levels of ROS drive NK cells to undergo robust cell division. This finding will elucidate a link between the levels of ROS and immune cell proliferation during viral infection and carcinogenesis.
Siglec-E negatively regulates the activation of Toll-like Receptor 4 by controlling its endocytosis
Authors: Chen Guoyun , wu Yin , Ren Dongren
Published: January 2023

TLR4 signaling is critical for providing effective immune protection but must be tightly controlled to avoid inflammation-induced pathology. Previously, we reported extensive and direct interactions between TLR and Siglec families of Pattern Recognition Receptors. Here, we examined the biological significance of this interaction during infection. We found that Siglec-E is required for E. coli-induced endocytosis of TLR4. Siglec-E-deficient dendritic cells infected with E. coli fail to internalize TLR4. This leads to sustained TLR4 on cell surface and activation of NF-κB and MAP kinase p38, resulting in high levels of TNF-α and IL-6 compared with wild-type dendritic cells. In contrast to the signaling events occurring at the plasma membrane, as a result of the inability to internalize of TLR4, Siglec-E-deficient dendritic cells were also defective for TRIF-mediated IFN-β production in response to E. coli infection. Furthermore, we found that accumulation of ubiquitinated-TLR4 and binding of E3 ubiquitin ligase Triad3A to TLR4 was increased significantly in bone marrow-derived dendritic cells from wild-type mice, but not from Siglec-E-deficient mice, after E. coli infection. This represents a newly discovered mechanism that regulates the signaling of TLR4 during E. coli infection.
C-reactive protein modulates myeloid derived suppressor cell function and signaling.
Authors: Jimenez Rachel V , Szalai Alexander J
Published: January 2023

Recently, we provided evidence that C-reactive protein (CRP) plays a causal role in acute kidney injury (AKI) i.e., human CRP exacerbates AKI in CRP transgenic mice (CRPtg) and AKI severity is lessened in CRP knockout mice. The CRP effect in CRPtg was associated with significantly increased renal infiltration by myeloid derived suppressor cells (MDSC). Subsequently, we showed that the AKI exacerbating influence of CRP in CRPtg could be blocked by depletion of MDSCs. In the present work we sought to define the impact of CRP on MDSC ontogeny and function. Using wild type mouse bone marrow (BM) we derived MDSCs and established that CRP enhances their ability to suppress CD3ɛ/CD28-stimulated T cell proliferation in vitro. By flow cytometry we verified that mouse BM-MDSCs express variable amounts of three CRP receptors: LOX-1, FcγRI, and FcγRIIB. Expression of the latter on BM-MDSCs was upregulated upon CRP exposure, as demonstrated by flow cytometry. Furthermore, preliminary data obtained from studies using BM-MDSCs derived from FcγRIIB deficient mice suggest that FcγRIIB participates in CRP-mediated signaling. In their sum, these data suggest that CRP-mediated augmentation of MDSC suppressive function is FcγRIIB-dependent. Ongoing work seeks to define the consequences of CRP-mediated modulation of MDSC functions in vivo and to explore whether FcγRIIB and other CRP receptors are involved.
ROCK2, but not ROCK1 interacts with STAT3 to regulate TH17/TFH gene transcription
Authors: Chen Wei , Nyuydzefe Melanie S. , Weiss Jonathan M. , Zhang Jingya , Waksal Sam D. , Zanin-Zhorov Alexandra
Published: January 2023

Rho-associated kinase 2 (ROCK2) was recently shown to be implicated in regulation of IL-17 and IL-21 secretion in both mice and humans via down-regulation of STAT3 phosphorylation and subsequent transcription activity. Here we report that the ROCK2, but not ROCK1 protein binds phosphorylated-STAT3 (pSTAT3) in both cytoplasmic and nuclear compartments in human CD4+ T cells during T helper 17 (TH17)-skewing activation. The interaction of ROCK2 with pSTAT3 was partially abrogated by treatment of cells with selective ROCK2 inhibitor suggesting that ROCK2 kinase activity is required for formation of JAK-STAT complex and STAT3 phosphorylation. Further analysis by chromatin-immunoprecipitation sequencing (ChIP-seq) revealed that ROCK2 binding is significantly enriched toward promoter regions and peaks at transcription start sites (TSS) relative to genomic DNA. Moreover, 60–70% of ROCK2 and STAT3 peaks are overlapped genome-wide and co-localized to several key genes that control TH17 and T follicular helper (TFH) cell functions. Specifically, the binding of ROCK2 and STAT3 to the IRF4 and Bcl6 genes was validated by ChIP-qPCR analysis performed in human CD4+ T cells activated by TH17-skewing conditions. Finally, siRNA-mediated inhibition of STAT3 attenuated ROCK2 binding to the IRF4 and Bcl6 promoters indicating the critical role of STAT3 in the recruitment of ROCK2 to chromatin and ROCK2-mediated regulation of TH17/TFH gene transcription. Together, the present work provides previously unidentified insights into the molecular mechanism of specific involvement of ROCK2 isoform in regulating STAT3 phosphorylation and transcriptional activity in TH17-driven autoimmune responses.
Helios plays a critical role in the effective differentiation of naive CD4+ T cells in vivo.
Authors: Skadow Mathias , Shevach Ethan M , Thornton Angela M
Published: January 2023

The transcription factor Helios is required for the maintenance of the suppressive phenotype of Foxp3+ regulatory T cells (Tregs) as evidenced by the spontaneous autoimmune activation that develops in mice with a Foxp3-specific Helios deficiency. Helios is also upregulated in a subset of activated CD4+Foxp3−T conventional (Tconv) cells, but its role in Tconv cells remains unknown. Mice with a CD4-Cre driven Helios deletion do not exhibit autoimmunity despite the defect in the suppressor function of their Treg population, pointing to a requisite function of Helios in the activation of Tconv cells in response to antigen. We examined the capacity of Helios-deficient, naïve Tconv cells to respond to their cognate antigen in vivo and found that Helios was not required for proliferation, differentiation into helper subsets, or the production of effector cytokines. Unexpectedly, we observed that Helios deficiency increased the susceptibility of naïve T cells to differentiate into Tregs under inflammatory conditions. Additionally, we found that Helios is necessary for the reactivation of memory T cells as Helios-deficient T cells failed to proliferate upon secondary challenge with antigen. Together, these findings suggest that increased Treg differentiation in the periphery and defective memory T cell reactivation in mice with a CD4-Cre driven Helios deletion may prevent the development of autoimmunity normally associated with Helios-deficient Tregs.
Novel microRNAs in the pathogenesis of rheumatoid arthritis
Authors: Singh Ram Pyare , Hahn Bevra H
Published: January 2023

Rheumatoid arthritis (RA) is a systemic inflammatory autoimmune disease with progressive joint destruction, lifelong disability, functional deterioration, and high mortality. The major effector cells in RA are T cells and synovial fibroblasts. Defects in T cells and synovial fibroblasts lead to activation of proinflammatory cytokines, which induce Th1 and Th17 cells and chronic inflammation. Recent studies provide further evidence of dysregulation of candidate microRNAs in inflammation and autoimmunity. In our preliminary studies we have found dysregulated candidate microRNA (miRNA21, miR25 miR95, miR124a, miR146a and miR186) expression in RA patients compared to healthy controls. Furthermore proinflammatory cytokines such as IL-6, IL-12, IL-17 and IL-21 are significantly increased in RA patients compared to healthy controls, and expression of these cytokines positively correlates with the expression of candidate microRNAs such as miR21 and negatively correlates with the expression of miR146a. Furthermore we found that miR21 and miR124a positively correlate, and miR146a negatively correlates, with DAS28 score in RA patients. Based on these preliminary data, we hypothesize that dysregulated microRNAs play an important role in RA pathogenesis. This study has translational potential and may identify novel therapeutic targets in patients with RA.
SOCS1-KIR peptide inhibition of lupus pathology is correlated to enhanced PD1/PDL1 expression
Authors: Collins Teresa D. , Mishra Shiwangi , Lam Brandon Krishna , Mohamed Zaynab Sidi , Veal Antia E. , Johnson Howard M. , Larkin Joseph
Published: January 2023

Lupus progression is driven by the accumulation of auto-inflammatory T cells producing cytokines such as interferon-gamma (IFN-γ). As such, the enhancement of co-inhibitory signaling pathways or retardation of inflammatory programming in T cells are potential strategies to ameliorate lupus pathologies. Notably, Suppressor of Cytokine Signaling-1 (SOCS-1) is a critical regulator of the Jak/STAT pathway, and pro-inflammatory cytokines associated with lupus. We have previously shown that a peptide mimicking SOCS1 signaling (SOCS1-KIR) inhibited IFN-γ signaling and auto-inflammatory disease progression. In this study, we test the hypothesis that SOCS1-KIR administration will inhibit lupus pathology in MRLlpr/lpr mice. A 3x/week administration of SOCS1-KIR mediated a statistically significant reduction in spontaneous skin lesions and the accumulation IFN-γ producing memory cells within the peripheral blood, spleen, and lymph nodes. Notably, SOCS1-KIR mediated decreases in lympho-accumulation were correlated to increased levels of the co-inhibitory molecules, PD1 and PD-L1 in vivo. In vitro interrogation revealed that SOCS1-KIR, but not a control peptide, mediated the upregulation of PD1 on CD8+ and CD4+ T lymphocytes in 5 days of culture. Moreover, SOCS1-KIR facilitated PDL1 surface expression on antigen presenting cells. Together, these data show that SOCS1-KIR treatment was effective in mitigating auto-inflammatory T cell accumulation and effector functions. Moreover, these studies suggest an interplay between SOCS1 function and PD1/PD-L1 signal regulation.
Mesenchymal stem cells ameliorate lupus symptoms in MRL/lpr mice by inhibiting T cell functions in a CCL2 dependent manner
Authors: Kim Ki Hun , Lee Hong Kyung , Lee Jae Hee , Kim Ji Sung , Choi Jeong Eun , Ji A Young , Hong Jin Tae , Kim Youngsoo , Han Sang-Bae
Published: January 2023

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the emergence of autoreactive lymphocytes, autoantibody production, and deposition of immune complexes in various tissues. Mesenchymal stem cells (MSCs) ameliorate SLE by inhibiting T cell functions, but the underlying mechanisms remain incompletely understood. Human MSCs increased the survival rates of lupus-prone MRL/lpr mice by inhibiting cytokine expression and kidney infiltration of T cells. In cellular assays, allogeneic MSCs inhibited the proliferation and the cytokine production of MRL/lpr-derived T cells. Using time-lapse imaging, we examined how MRL/lpr-derived T cells gather information from MSCs at the single-cell level. Wild type MSCs demonstrated induction of T cell migration, and long-term contact duration than CCL2-knockdowed/knockout MSCs. MSC-driven CCL2 increased the expression of vascular cell adhesion molecule-1 expression in MRL/lpr-derived T cells. Taken together, our study helps to understand how MSCs inhibit autoreactive T cell functions by using CCL2 and ultimately how MSCs ameliorate symptoms in lupus-prone MRL/lpr mice.
LXR and RORG modulate bi-directional, sumoylation-dependent pathways in chronic autoimmune diseases
Authors: Chang Mi Ra , Strutzenberg Timothy , Griffin Patrick Robert
Published: January 2023

The nuclear receptor RORG (RAR-related orphan receptor gamma; NR1F3) plays an essential role in the development of T helper cells leading to the production of interleukin-17. Previously, we reported that the potent and selective RORG inverse agonist SR2211 was effective at repressing RORG activity in cultured cells and also demonstrated robust efficacy in a murine model of arthritis, the collagen induced arthritis (CIA) model. Lead optimization of the SR2211 chemical scaffold lead to the identification of compounds that maintain potent RORG-specific inverse agonism while exhibiting modest LXR activation. One such compound, SR1903, demonstrated improved efficacy in the CIA model with significantly improved therapeutic index. Proteogenomic analysis revealed that SR1903 increased expression of SENP3 (SUMO1/Sentrin/SMT3 specific peptidase 3) resulting in SUMOylation-dependent, opposing modulation of RORG and LXR activities upon inflammatory stimulation of T cells. Ongoing mutational studies and receptor trafficking analysis will provide additional mechanistic insight into this novel inflammatory process particulary in the context of NCoR tethering to promoters of RORG and LXR target genes.
V1V2 multivalent scaffolds induce focused antibody responses with functional activity, prolonged durability, and modest neutralization potency
Authors: Hessell Ann Jones , Jiang Xunqing , Totrov Max , Powell Rebecca , Hioe Catarina , Haigwood Nancy L. , Kong Xiang-Peng , Zolla-Pazner Susan
Published: January 2023

The V1V2 region of HIV-1 gp120 is vulnerable to a group of broadly neutralizing monoclonal antibodies (bnMAbs), and RV144 vaccinees developed strongly binding Abs to this region that correlated with a reduced risk of infection. This region of Env is now recognized as an important vaccine target, notwithstanding its relatively poorly immunogenic epitopes. We hypothesized that presenting V1V2 in multivalent scaffolds could improve immunogenicity and structurally constraining the epitopes could induce a more focused immune response in animal models. We designed of a panel of immunogens engrafting the V1V2 domain into trimeric and pentameric scaffolds. Here, we report results of immunogenicity studies in nonhuman primates (NHP) with 2 different V1V2 trimeric scaffolds. Our experimental approach in NHP consisted of 3 DNA and protein co-immunizations and a single protein boost. The DNA components consisted of 50μg of ZM109 gp120 plasmid delivered by gene gun with a simultaneous intramuscular injection of 100 μg of V1V2 scaffold protein immunogens. NHP immune responses were specific for V1V2 conformational epitopes with cross-reactive binding to heterologous gp120s and were functionally active in phagocytosis assays. Notably, V1V2 binding Ab responses were detectable 2 years after immunizations ceased. Neutralizing Abs developed in vaccinated NHP to Tier 1A and 1B, Clade B and Clade C viruses. The V1V2 structurally constrained scaffolds show promise as novel and highly effective at inducing functional antibody responses specifically targeted to the epitopes presented. Our results advance pre-clinical data in support of developing V1V2 vaccine candidates despite the structural complexity of the region.

Funding: P01AI100151-03
Antibody epitopes identified in critical regions of dengue virus nonstructural 1 protein in mouse vaccination and natural human infections
Authors: Hertz Tomer , Beatty Robert P , MacMillen Zachary , Killingbeck Sarah S , Wang Chunling , Harris Eva
Published: January 2023

A major challenge in dengue vaccine development is that cross-reactive anti-DENV antibodies (Abs) can be protective or potentially enhance disease via antibody-dependent enhancement (ADE). We recently showed that immunization with adjuvanted recombinant NS1 from all four DENV serotypes protects against DENV2 challenge in a mouse model of lethal vascular leak syndrome. Conversely, we found that DENV NS1 by itself triggers endothelial permeability in vitro and vascular leak in vivo. Here, we evaluated survival to lethal DENV challenge in mice immunized with (1) NS1+alum; (2) NS1+Monophosphoryl Lipid A and AddaVax (MA); (3) NS1+Sigma adjuvant system and CpG DNA (SCpG); (4) ovalbumin+adjuvant (OVA); or (5) a sublethal dose of DENV2 strain PLO46.NS1. OVA or NS1+alum immunized mice were not protected, whereas immunization with NS1+MA or NS1+SCpG or DENV2-PL046 resulted in 100% survival. We characterized Ab responses to NS1 from DENV1-3 using an antigen microarray tiled with 20-mer peptides overlapping by 15 amino acids and identified 5 regions with significant levels of antibody reactivity in the NS1+MA group. We also profiled Ab responses to NS1 in humans naturally infected with DENV-2 or -3 in longitudinal serum samples from Nicaragua. One region in the NS1 “wing” domain was immunodominant in both mouse and human studies, and two regions were identified only in NS1-immunized mice; thus, vaccination can generate Abs to regions that are not targeted in natural infection and could provide additional protection against a lethal DENV infection. Overall, we identified several immunodominant regions, which were in functionally important locations on the DENV NS1 protein and are potential correlates of protection.
Intradermal Delivery of INO-4201 Drives Robust Ebola Glycoprotein Specific Humoral and Cellular Immune Responses in Healthy Volunteers in an Open Label Phase I Trial
Authors: Kraynyak Kimberly Ann , Amante Dinah , Tebas Pablo , Knoblock Dawson , Sylvester Albert J , Morrow Matthew P , Prostak Naseem , Liu Li , Sakata Lindsay , Cheung Jocelyn , Desfor Shane , Patel Ami , Dallas Michael , Yan Jian , Khan Amir , Boyer Jean D , White Scott , Maslow Joel , Humeau Laurent , Sardesai Niranjan , Weiner David , Bagarazzi Mark
Published: January 2023

The most recent Ebola outbreak in West Africa resulted in over 28,000 suspected cases of infection and over 11,000 deaths. As no specific immune correlate has been associated with protection against Ebola to date, the development of a vaccine that generates robust humoral and cellular immune responses may be the best approach to achieve full protection. INO-4201 is a plasmid-based prophylactic vaccine targeting Zaire Ebola glycoprotein (GP), designed to prevent Ebola infection. INO-4201 encodes a consensus antigen that encompasses genetic variability from previous outbreak strains to broaden immune coverage for divergent Ebola virus variants. Intradermal (ID) administration of a 2 or 3-dose regimen of INO-4201 followed by in vivo electroporation with the CELLECTRA® device was well tolerated in 140 healthy volunteers with no related Grade 3 or Grade 4 SAEs reported. INO-4201 induced robust Ebola GP-specific antibody and T cell responses in the healthy volunteers. The majority of patients seroconverted, as gauged by binding ELISA after only 2 doses of INO-4201. EBOV GP specific T cell response were assessed by Interferon gamma (IFNγ) ELISpot revealing a mean peak response magnitude of 295 SFU per 106 PBMCs. Intracellular cytokine staining indicated that immunization with INO-4201 drove statistically significant increases in the production of IFNγ or Tumor Necrosis Factor alpha in both the CD8+ T cell compartment (p=0.001) and CD4+ T cell compartment (p=0.004). ID administration of INO-4201 using the CELLECTRA® device was well tolerated and immunogenic as assessed by both humoral and cellular EBOV GP-specific immunoassays supporting INO-4201 as a strong candidate for further clinical development of a prophylactic Ebola vaccine.
Transcription of immune genes at the fetomaternal interface during pregnancy in a model marsupial, Monodelphis domestica.
Authors: Hansen Victoria L , Miller Robert D
Published: January 2023

The evolutionary challenge of nourishing an allogeneic conceptus in the presence of an adaptive immune system has intrigued immunologists since Sir Peter Medawar brought attention to the “fetal allograft problem” in the 1950s. Though much has been learned in reproductive immunology, it has largely been limited to eutherian mammals such as humans, mice, and mammalian livestock. Therians are the lineage of mammals that include both eutherians, or “placentals”, and marsupials. Though eutherians and marsupials diverged approximately 148 million years ago, the two lineages share a common viviparous ancestor. Therefore marsupials and eutherians likely share immunological traits during pregnancy. Previous RNA-Seq studies demonstrated increased transcription of immune genes in late, terminal pregnancy. However since the RNA-Seq data only represented one pregnancy time point, less than 24 hours until birth, it was unclear whether immune gene transcription should be attributed to pregnancy, implantation, or parturition. We expanded time points to target the last 144 hours of pregnancy during which both implantation and parturition occur in Monodelphis domestica. We examined the transcription of cytokines as well as complement and their regulators at the fetomaternal interface in pregnant opossums. We found a significant increase in transcription of cytokines in the last 24 hours of pregnancy and comparatively little transcription of cytokines at the time of implantation. There was also little transcription of some complement components throughout pregnancy, particularly those in the upstream cascade. Some downstream complement components trended toward increased transcription in the last 48 hours of pregnancy.
Interaction of γδ TCR with the WC1 hybrid co-receptor/pathogen recognition receptor in cattle.
Authors: Gillespie Alexandria , Connelley Timothy , Telfer Janice C , Baldwin Cynthia L
Published: January 2023

Ruminants have a larger proportion of γδ T lymphocytes in blood compared to mice and humans. These lymphocytes are first responders to pathogens such as Leptospira and Mycobacterium. Bovine γδ T cells that respond to these pathogens express members of a unique γδ T-cell specific molecular family WC1 that are hybrid pattern recognition receptors and signaling co-receptors. Only γδ T cells that express WC1 molecules that bind to the pathogens selectively respond by proliferation and cytokine production. WC1 molecules have also been shown to augment signaling when co-crosslinked with the TCR but cannot signal on their own. Silencing WC1 through shRNA results in inhibition of the response to leptospira. Together these data indicate that WC1 is crucial for activation of the cells and we wished to further understand how the TCR interacts with WC1. The TCR is transcribed from a recombination of variable, joining, and diversity genes resulting in hundreds to millions of different possible sequences while the WC1 family has only 137 different pathogen-binding SRCR domains coded for by 13 genes. Yet only γδ T cells that express particular WC1 family members respond to leptospira suggesting it has primacy. We hypothesize that WC1 molecules co-localize with the TCR following binding of the pathogen. To test this hypothesis we used Imaging flow cytometry. Using FRET analysis we are able to determine that not only are the TCR and WC1 co-localized in the synapse following activation but they also are interacting closely within 9nm of each other. To evaluate the role of the TCR in dictating specificity of the response we are using next generation sequencing of transcripts coding for the TCR gamma and delta chains in leptospira-responsive cells.
Comparison genomics of two closely related isolates of Clostridium perfringens full genome sequences reveals regions of genome plasticity with prevention potential
Authors: Li Charles Z , Yan Xianghe , Lillehoj Hyun S.
Published: January 2023

The spore-forming anaerobic Clostridium perfringens (CP) is the primary etiological agent of necrotic enteritis (NE) disease, one of priority enteric diseases in chickens which is responsible for annual losses of $6 billion in the US poultry industry. Our long term goal is to develop a recombinant vaccine against NE as an antibiotic alternative. Because the design of effective vaccines requires a thorough understanding of the pathogen’s entire genome and their functions, we sequenced the genomes of two closely related Type A CP strains: Del1 and CP15, isolated from NE-afflicted chicken farms. Del1 is very virulent, while CP15 produced very mucoid supernatant in its growth medium culture. PacBio and Illumina sequencing technologies were used to generate accurate de novo and hybrid genome assemblies. In this study, these two assembled full genomes are deposited into NCBI GenBank, annotated and analyzed. Del1 has a circular genome of 3.55 M base pairs (bp) long and four plasmids (82326, 69601, 49550, and 49456 bp) with a total of 3361 predicted open reading frames. CP15 has a genome of 3.34 M bp long and four plasmids (14755, 3843, 3202 and 2421 bp) with a total of 2997 predicted CDS. The sequenced data revealed that Del1 contains a pore-forming toxin netB encoded within one of highly conserved NE-associated loci. In contrast, CP15 did not contain netB gene. Sequenced data also showed that Del1 has a unique ribosomal protection protein which protects ribosomes from the translation inhibition of tetracycline (YP_001967743). These results provide important insight into the pathogenesis of CP in avian NE and novel information on potential targets of toxins and virulence factors that will facilitate the vaccine design.
Missense variant in XYZ results in hyperactivation of mTOR signaling pathway and increased T-cell senescence
Authors: Raquer Hayley M , Fritz Jill , Lenardo Michael
Published: January 2023

Phosphatidylinositol-4-phosphate 5-kinase I (XYZ) is a member of the class I PIP5K family that is responsible for the phosphorylation of phosphatidylinositol 4-phosphate (PIP) to produce phosphatidylinositol 4,5-biphosphate (PIP2). PIP2 is an important intermediate in the PI3K signaling pathway and is phosphorylated by PI3K to create phosphatidylinositol 3, 4, 5-triphosphate (PIP3), which is an important signaling molecule in TCR activation. Through whole exome sequencing, we have recently identified a 14-year old female Turkish patient with a homozygous missense variant in the activation loop of XYZ. The patient presents with lymphadenopathy, hemolytic anemia, low IgA, no IgE, positive antinuclear antibodies, and reactive airway disease. She has low naïve T-cells, increased CD45RA+ T-effector memory (TEMRA) cells, and increased CD57, a marker of T-cell senescence, on CD8+ T-cells. Patient T-cell blasts show increased phosphorylation of S6 levels upon TCR stimulation. Further, overexpression of mutant XYZ in T-cells resulted in increased phosphorylation of S6 at baseline compared to overexpression of wild type XYZ. Overall, these results suggest that the XYZ variant leads to hyperactivation of the mTOR signaling pathway, which may contribute to T-cell senescence and disease.

This research was supported [in part] by the Intramural Research Program of the NIH, NIAID
Defective activation of IL2-inducible T cell kinase (ITK) in XMEN disease
Authors: Kanellopoulou Chrysi , George Alex B , Masutani Evan , Jiang Ping , Ravell Juan C , Chaigne-Delalande Benjamin , Lenardo Michael
Published: January 2023

We have previously described a primary immunodeficiency due to mutations in the magnesium transporter MAGT1, named XMEN disease (X-linked Magnesium defect with Epstein-Barr infection and Neoplasia). Loss of MAGT1 results in impaired T cell receptor signaling. Previous analyses suggested that proximal TCR signaling was unaffected in XMEN patients but PLCg1 phosphorylation was delayed. We have further characterized the TCR activation defect in MAGT1 deficient cells and found that the primary cause is the impaired activity of Interleukin-2 inducible T cell kinase (ITK). ITK is phosphorylated normally by LCK and recruited to immunological synapse but fails to phosphorylate downstream targets efficiently. A similar phenotype is apparent when cells from normal controls are activated in the absence of extracellular magnesium. A transient influx of magnesium is sufficient to restore ITK activity and PLCg1 phosphorylation in XMEN T cells. We are in the process of further characterizing the molecular underpinnings of ITK regulation by MAGT1.

Acknowledgements:

This research was supported by the Intramural Research Program of NIAID, NIH
Recruitment of calcineurin to the T cell receptor positively regulates T cell activation
Authors: Dutta Debjani , Barr Valarie A , Akpan Itoro , Mittelstadt Paul R , Singha Laishram Indira , Samelson Lawrence E , Ashwell Jonathan D
Published: January 2023

Calcineurin is a phosphatase whose primary targets in T cells are NFAT transcription factors, and its inhibition with cyclosporin A (CsA) or FK506 is a cornerstone of immunosuppressive therapies. Here we show that calcineurin is recruited to the T cell receptor signaling complex where it reverses the inhibitory phosphorylation of Lck on Ser-59 (LckS59). Loss of calcineurin activity impaired phosphorylation of ZAP-70Y493 and some downstream pathways in a manner consistent with signaling in cells expressing LckS59A (non-phosphorylatable) or LckS59E (phosphomimetic). Importantly, CsA inhibited activation-induced LFA-1-dependent and NFAT-independent adhesion of T cells to ICAM-1, with little effect on cells expressing mutant Lck. These results provide a new understanding of how widely-used immunosuppressive drugs interfere with essential processes in the immune response.
Testing the susceptibility of mice with normal microbial experience to induction of allergic airway disease
Authors: Block Katharine E , Masopust David , Kita Hirohito , Hamilton Sara E , Jameson Stephen C
Published: January 2023

Allergic asthma is a large and growing medical problem in the developed world. The hygiene hypothesis posits that improved sanitation, vaccination programs, and increased antibiotic use have limited our exposure to natural infections, leading to immune systems that are more vulnerable to pathological responses. It has been difficult to test whether these advancements in public health can explain some of the increased incidence in allergic asthma, partly because laboratory mice commonly used in research are housed in a clean and highly controlled environment (known as specific pathogen free, or SPF). Cohousing SPF laboratory mice with mice from pet stores leads to acquisition of natural mouse pathogens through physiological animal-to-animal transmission. Our research group has demonstrated that this normal microbial experience dramatically alters the immune system, making it more similar to the immune system of adult humans. We used this “dirty” cohousing mouse model to test the effect of physiological microbial exposure on susceptibility to allergic airway disease. SPF and cohoused mice were sensitized and challenged intranasally with house dust mite (HDM) extract. Exposure to HDM induced eosinophilic airway inflammation and immune cell infiltration in SPF mice that resembles airway pathology of asthmatic patients. Serum levels of IgE and IgG1 antibodies to HDM were also elevated. Immune cell populations infiltrating the lung and activation of antigen-specific CD4+ T cells were also analyzed. We are currently investigating the immune response to this allergen exposure model in cohoused mice. Results from these studies will help explain the impact of environmental and microbial experience on susceptibility to allergic asthma.
Scavenger receptor class B type I (SR-BI) modulates glucocorticoid mediated lymphocyte apoptosis in asthma
Authors: Reece Sky W. , Kilburg-Basnyat Brita , Madenspacher Jennifer H. , Luo Bin , Capen Amanda , Fessler Michael B. , Gowdy Kymberly M.
Published: January 2023

Asthma is a heterogeneous airway disease that has increased significantly in recent decades. Clinical studies have shown a positive correlation between serum large high-density lipoprotein (HDL) levels and airway protective effects in asthma. However, how cholesterol communicates with the lung during asthma is still not well understood. Scavenger receptor class B type I (SR-BI) is an HDL receptor that has also been shown to regulate glucocorticoid production. SR-BI has also been shown to regulate lymphocyte apoptosis in models of sepsis and atherosclerosis. Recently, we reported that SR-BI dampens the pulmonary innate immune response, but the role of SR-BI in asthma is unknown. Therefore, SR-BI+/+ and SR-BI−/− mice were sensitized with 10 μg house dust mite (HDM) or PBS by oropharyngeal aspiration on day 0, 7, and challenged with 2 μg HDM on day 14, 15, 16. Airway inflammation, cytokines, and serum corticosterone levels were quantified on day 17. SR-BI−/−mice had increased pulmonary neutrophils and lymphocytes after HDM challenge when compared to SR-BI+/+ mice. SR-BI−/− mice had consistently lower levels of serum corticosterone after HDM or PBS exposure. To determine the role of glucocorticoid production in asthma, SR-BI+/+ and SR-BI−/− mice were supplemented with corticosterone in their drinking water during HDM exposure. SR-BI−/− mice with corticosterone treatment had a decrease in pulmonary lymphocytes after HDM exposure. The observed decrease in lymphocytes may be due to glucocorticoid induced apoptosis which was absent in SR-BI−/− mice not treated with glucocorticoids. Our study has shed new light on how cholesterol receptors such as SR-BI play an important role in glucocorticoid mediated lymphocyte apoptosis in asthma.
Age-associated changes in intrathymic B cell function
Authors: Cepeda Sergio , Cantu Carolina , Xiao Yangming , Griffith Ann
Published: January 2023

Autoimmune disease affects approximately 3% of the U.S. population and is the leading cause of death among young and middle-aged women. Susceptibility to autoimmune disease generally increases with age in both sexes; however, mechanisms linking aging and increased susceptibility are incompletely understood. One hallmark of the aging immune system is atrophy of the thymus, the primary site of T lymphocyte generation. We recently showed that in addition to size, critical thymic functions are lost with age, including expression of tissue restricted self-antigen (TRA) genes. Thymic TRA expression allows clonal deletion of potentially auto-reactive T cells, and loss of TRA expression during aging represents a potential mechanism for age-associated increases in autoimmunity. Until recently, only epithelial cells were known to express TRAs within the thymus, but it is now clear that IgM-IgD-intrathymic B lymphocytes can also be induced to express Aire (Autoimmune Regulator), a factor critical for TRA expression, as well as downstream Aire-dependent TRAs. Here, we find that B cell frequency increases 5-fold in the aged murine thymus, concomitant with a shift from an IgM-IgD-to an IgM+IgD+ phenotype. Using next-generation RNA sequencing, we show significant decreases in Aire and TRA gene expression in aged intrathymic B cells. Our findings indicate that age-associated changes in intrathymic B cells inhibit their ability to negatively select auto-reactive T cells, revealing a novel potential mechanism linking aging with increased susceptibility to autoimmune disease.
Identification of novel plasma cell subsets in a mouse model of SLE
Authors: Rosean Timothy R , Loo William , Erickson Loren D
Published: January 2023

Systemic lupus erythematosus (SLE) is an autoimmune disease driven by the secretion of anti-nuclear antibodies (ANA) from a subset of terminally differentiated B cells, called plasma cells (PC). PC persist in the bone marrow for the lifetime of the host, posing challenges for the development of therapies for SLE and other autoimmune diseases. The mechanisms of long-term persistence of PC are poorly understood. Utilizing the congenic New Zealand Black Autoimmunity 2 (NBA2) mouse strain, which develops IgG ANA similar to SLE, our lab has identified novel subsets of mature bone marrow PC. which are highly heterogeneous in both phenotype and function. We have characterized these populations of PC based on the expression of CD93 and CXCR3. While CD93+ PC are largely quiescent, CXCR3+ PC are highly proliferative in the bone marrow. In addition, CXCR3+ PC are highly enriched for IgG secretion, while CD93+ PC are enriched for IgA secretion. CD93+ PC are detected in the bone marrow of B6 mice at early time points after immunization with KLH, whereas CXCR3+ PC are nearly absent in the bone marrow yet are present in the spleen. In contrast, significant numbers of both CD93+ and CXCR3+ PC are found in both the spleen and bone marrow of NBA2 mice. Characterization of transcription factors reveals that all PC populations express high levels of BLIMP-1 and IRF-4, demonstrating their commitment to a mature PC fate. These data suggest that distinct subpopulations of PC may be responsible for the pathogenesis of SLE. Current research is focused on elucidating the mechanism by which distinct PC subsets give rise to ANA.
The role of the immunomodulatory enzyme indoleamine 2,3-dioxygenase 2 (IDO2) in initiation, development, and treatment of autoimmune disorders
Authors: Merlo Lauren MF , Grabler Samantha , DuHadaway James B , Manley Kaylend , Prendergast George C , Muller Alexander J , Laury-Kleintop Lisa D , Mandik-Nayak Laura
Published: January 2023

The tryptophan catabolizing enzymes indoleamine 2,3-dioxygenase (IDO) 1 and 2 lie at the interface between metabolism and immunity. Recently, we identified a unique functional role for IDO2 in modulating the initiation and severity of autoimmune arthritis in a murine model of disease. We find that IDO2, but not its better-studied counterpart IDO1, acts as a pro-inflammatory mediator affecting autoantibody production and T helper cell function in the KRN preclinical model of arthritis. Reciprocal adoptive transfer experiments demonstrate that IDO2 acts by a B-cell intrinsic mechanism to regulate inflammation. IDO2 function in B cells was contingent on a cognate, antigen-specific interaction to exert its immunomodulatory effects on arthritis development. Alterations in costimulatory molecules and associated cytokines involved in cross-talk between B and T cells suggest that IDO2 acts at the T:B cell interface to modulate the potency of T cell help needed to promote autoantibody production. Given the paucity of novel therapies for rheumatoid arthritis and related autoimmune disorders, IDO2 should be considered as a potential novel therapeutic target for modulating disease pathways leading to autoimmunity. However, therapeutically targeting IDO2 has been challenging due to the lack of small molecules that specifically inhibit IDO2 without also affecting the closely related IDO1. To this end, we have recently begun development and characterization of approaches that ameliorate disease by specifically targeting IDO2. We find that we are able to recapitulate the reduction in arthritis seen in genetic knockouts with IDO2-targeted therapies in preclinical models of disease.