bluuebunny/crossref_metadata_2025 huggingface dataset:
Glutathione S-transferases as regulators of tumor-induced myeloid-derived suppressor cell survival (66.38)
Authors: Fitzgerald Phillip , Beury Daniel , Ostrand-Rosenberg Suzanne
Published: January 2023

Tumor-induced myeloid-derived suppressor cells (MDSC) are a major barrier to tumor immunotherapy because they inhibit T-cell anti-tumor immunity through various mechanisms, including cystine sequestration and production of reactive oxygen species (ROS). MDSC accumulation, suppressive potency, and survival are driven by inflammation, which also increases MDSC production of ROS. Surprisingly, ROS do not adversely affect MDSC, suggesting that MDSC neutralize endogenous ROS. Because MDSC survival is likely to be controlled by the mechanisms that protect them against endogenous ROS, we are identifying molecules that regulate ROS levels with the goal of identifying potential targets for inducing MDSC apoptosis. Glutathione (GSH) is the major antioxidant that detoxifies ROS in conjunction with various isoforms of GSH S-transferase (GST) that catalyze the detoxification process. Because of their role in detoxifying intracellular ROS, we are analyzing intracellular levels of GSH and multiple GST isoenzymes in conventional and inflammatory MDSC to understand how inflammation protects MDSC and thus promotes tumorigenesis. GST isoforms that facilitate MDSC survival will be novel drug targets for reducing tumor-induced immune suppression and facilitating tumor immunotherapy.
Key role played by a butyrophilin family gene product in human gd T cell activation (66.39)
Authors: Bonneville Marc , Harly Christelle , Guillaume Yves , Olive Daniel , Scotet Emmanuel
Published: January 2023

The major peripheral Vg9Vd2 T cell subset in humans is activated by small phosphorylated metabolites of the isoprenoid synthesis pathway (referred to as phosphoantigens (P-Ag)), which are overexpressed in tumor and infected cells. The molecular partners and the mechanisms involved in P-Ag-mediated activation of Vg9Vd2 T cells remain elusive. In line with the recently described implication of murine Skint-1, a butyrophilin (BTN) family-related molecule, in selection of murine gd T cells, we show that several mAbs against a human BTN-related product with yet unknown function, which is ubiquitously expressed at the surface of hemopoietic cells sensitize a broad array of target cells to specific Vg9Vd2 T cell recognition (cytolysis, cytokines release, proliferation). Although this process is Vg9Vd2 TCR-dependent and involves gamma/delta chain residues previously shown to be critical for P-Ag-induced activation, we failed to detect any significant modulation of MVA pathway genes expression, nor any increase of endogenous P-Ag (IPP/ApppI) accumulation within mAb-treated target cells. We also identified a BTN-specific mAb that specifically inhibited Vg9Vd2 T cell activation triggered by endogenous or exogenous P-Ag.Moreover BNT knockdown abrogated Vg9Vd2 responses to PAg. Taken together, our results support a unique and critical contribution of BTN-related products in the regulation of TCR-mediated activation of human Vg9Vd2 T cells.
Downstream targets of PKCβII in dendritic cell differentiation (66.4)
Authors: Spangler Haley , Carlson Louise , Lee Kelvin
Published: January 2023

Dendritic cells (DC) are antigen presenting cells, inducing both innate and adaptive immune responses. In a tumor setting their differentiation from progenitor cells is inhibited by tumor derived factors, resulting in an accumulation of immature DCs. When myeloid-derived DC differentiation is inhibited, these immature DCs act as myeloid derived suppressor cells (MDSCs) which support tumor growth and metastasis, as well as reduce efficacy of many therapeutic agents in cancer. Our lab has shown in humans that loss of PKCβII (a member of the PKC family of signal transduction molecules) expression blocks DC differentiation and that tumors may mediate this downregulation. It is also known that interferon regulatory factor 8 (IRF8) plays an important role myeloid cell differentiation from progenitor cells, and it is downregulated in tumor environments, inhibiting DC differentiation. In human and murine models, we determined that inhibition of PKCβII with CGP 53353 prevented differentiation of lineage negative progenitor cells to DCs as evaluated by DC phenotypic markers and allogeneic T-cell induction. We also found that PKCβII inhibition lead to a loss in IRF8 expression. Therefore we have demonstrated a role for PKCβII in DC differentiation in mice, as well as identified a line between IRF8 and PKCβII signaling during differentiation.
Differential T cell suppression through regulatory T cells (66.43)
Authors: James Edd , Bailey Ian , Reeves Emma , Elliott Tim
Published: January 2023

Depletion of regulatory T cells in the CT26 murine tumour model stimulates a robust protective T cell response which is also protective to challenge with other tumours of different histological origins. We have characterised a cross-protective antigen, GSW11 (GGPESFYCASW) presented by H-2Dd. This antigen was found to be encoded within the env (gp90) gene of an endogenous retrovirus emv-1 (MuLV), previously shown to encode other CT26 antigens. Further characterisation of GSW11-specific T cell responses has shown them to be suppressed by Tregs and are absent in CT26 challenged mice. In comparison, AH1-specific T cells (AH1 is a previously characterised CT26 antigen) are not suppressed. Here we show that the GSW11-specific T cell responses are present in CT26 challenged Treg replete mice for a limited period in the tumour of challenged mice. Characterisation of the AH1 and GSW11-specific T cells infiltrating the CT26 tumour reveal differences in expression of several molecules associated with cell survival and activation. These expression profiles provide an intriguing explanation for the differential suppression of tumour-specific T cells in vivo.
Combination therapy with dendritic cell based vaccination and myeloid derived suppressor cell depletion augments immunity in a murine pancreatic carcinoma model (66.8)
Authors: Pilon-Thomas Shari , Hall MacLean , Vohra Nasreen , Sarnaik Amod , Ghansah Tomar
Published: January 2023

Pancreatic cancer is an aggressive disease with poor prognosis. Immunotherapy may be less effective in treating pancreatic tumors due to suppression by myeloid derived suppressor cells (MDSC). In this study, we sought to determine the effect of combining MDSC depletion with dendritic cell (DC) vaccination in a murine model of pancreatic cancer. In mice, MDSC are characterized by surface expression of CD11b and Gr-1. After injection of Panc02 tumor cells, a higher percentage (29.2%) of MDSC were present in spleens of tumor-bearing mice as compared to naïve mice (6.38%, p<0.05). MDSC isolated from tumor-bearing mice significantly suppressed proliferation of antigen stimulated T cells. It has been reported that Gemcitabine (Gem), a chemotherapeutic drug, can selectively target MDSC. Flow cytometry results showed a decrease in the percentage of MDSC in splenocytes from Gem-treated tumor-bearing (6.57%) compared to untreated mice (24.3%, p<0.01). To study the effects of DC vaccination combined with MDSC depletion on tumor rejection, mice received PANC02 tumor cells and were treated with 3 injections of DC at 3-4 day intervals. After the final vaccination, mice received Gem. Survival was measured in 50% of mice receiving both DC vaccination and Gem (p<0.05 as compared to each treatment alone). Results from this study suggest that the depletion of MDSC enhances DC induced immunity in a murine model and raises the potential for this strategy in patients with pancreatic cancer.
A novel mouse model for arenavirus hemorrhagic fever (67.20)
Authors: Schnell Fred , Mourich Dan , Iversen Patrick
Published: January 2023

The viral family Arenaviridae includes a number of viruses that can cause hemorrhagic fever. Arenavirus infection often involves multiple organs and can lead to capillary instability, impaired hemostasis, and death. There is a lack of treatment options available to those suffering from arenaviral hemorrhagic fever and a well established mouse model of arenaviral hemorrhagic fever would benefit preclinical testing for arenavirus antivirals or therapeutics. We have identified the FVB mouse strain, which succumbs to a hemorrhagic fever like illness when infected with lymphocytic choriomeningitis virus (LCMV). FVB mice infected with LCMV demonstrate high mortality associated with thrombocytopenia, hepatocellular and splenic necrosis, and cutaneous hemorrhage whereas C57BL/6 mice survive. Investigation of possible inflammatory mediators revealed increased IFN-g and IL-6, along with increased chemokine production, at early times after LCMV infection as compared to C57BL/6 mice. Removal of CD4+ or CD8+ cells from FVB mice at time of infection prevented mortality in all treated animals and treatment with FK506 early during infection delayed disease onset. This report offers a novel animal model for arenavirus research and pre-clinical therapeutic testing.
TLR7-mediated Type I and Type III interferon production and TLR7 movement within primary human plasmacytoid dendritic cells is stimulated by Influenza virus (67.22)
Authors: Natalia Mahwish , Singh Sukhwinder , Cui Chongwei , Fitzgeral-Bocarsly Patricia
Published: January 2023

Human plasmacytoid dendritic cells (pDC) recognize single stranded RNA and double stranded DNA viruses through endosomal TLR7 and -9, respectively, resulting in production of type I and III interferons (IFN). pDC produce both IFN-α and IFN-λ upon Influenza (Flu) virus stimulation; this induction of IFN is dependent on signaling through TLR7 since the TLR7 inhibitor, IRS661, inhibited Flu-induced IFN production in pDC, but not HSV-induced IFN, which signals through TLR9. Cathepsins (Cat), cysteine endolysosomal proteases, are essential for antigen processing in flu-stimulated murine DCs. We found that the broad Cat inhibitor, Z-FM-FMK, inhibited flu and HIV-induced IFN-α production by pDC, both which require processing to make their RNA accessible to TLR7, but had no effect on the small molecule TLR7 ligand Imiquimod-induced IFN production. Flu is known to enter cells via the endocytic pathway, with viral membrane fusion with the endolysosomal membrane required for the release of its viral RNA into the cytoplasm and its subsequent replication. Using ImageStream imaging flow cytometry, we observed that TLR7 is constitutively present in the EEA1 (early)- and LAMP1 (late)-positive endosomes in pDC, and, upon stimulation with flu, TLR7 co-localization with LAMP1-positive endosomes increases. Taken together, these results indicate that recognition of Flu in pDC requires virus endocytosis, Cat processing, and results in TLR7 movement from early to late endosomes and IFN production.
Contribution of 2009 pandemic swine-origin influenza virus gene segments to virulence and immunity (67.23)
Authors: Stambas John , Evans Justin , Middleton Deborah , Lowther Sue , Robinson Rachel , Haining Jessica , Payne Jean , Marsh Glenn , Laurie Karen , Bean Andrew , Stewart Cameron , Gilbertson Brad
Published: January 2023

Influenza A viruses cause annual epidemics and occasional global pandemics as seen in 1918, 1957, 1968 and most recently in 2009. On the 11th of June 2009 the first global pandemic of the 21st century was declared as a result of a triple reassortant virus containing gene segments from circulating swine, avian and human influenza viruses. Reverse genetics technology was used to study swine-origin influenza virus (S-OIV) virulence through the generation of reassortant influenza viruses expressing gene segments from the 2009 pandemic strain. Our results show that S-OIV gene segments are capable of altering virulence in both X31 and PR8 reassortant influenza viruses. In particular one specific X31-(S-OIV) reassortant virus showed a significant increase in virulence in mice with substantial weight loss, high virus titers and elevated levels of inflammatory mediators in the lung following infection. Moreover these findings suggest that the current H1N1 pandemic S-OIV has the potential to influence the virulence of other circulating influenza viruses in the future.
Proteolytic processing of TNF-α by influenza-specific CD8+ T cells is required for alveolar epithelial cell MIP-2 production and severe lung injury in a transgenic mouse model of influenza infection (67.3)
Authors: DeBerge Matthew , Enelow Richard
Published: January 2023

Adoptive transfer of influenza hemagglutinin (HA)-specific CD8+ T cells into transgenic mice expressing influenza HA on the alveolar epithelium results in epithelial cell production of monocyte chemotactic protein (MCP)-1 and macrophage inflammatory protein (MIP)-2, the subsequent mononuclear cell infiltration and diffuse alveolar damage. The lung injury observed in this model is mediated by CD8+ T cell production of tumor necrosis factor (TNF)-α. TNF-α is expressed as a transmembrane (tm) protein that is proteolytically processed to produce soluble (s) TNF-α. To determine the roles of tmTNF-α and sTNF-α in CD8+ T cell mediated lung injury, we generated HA-specific CD8+ T cells that only express a noncleavable tmTNF-α (TM). We found that proteolytic processing of tmTNF-α to sTNF-α was required for alveolar epithelial cell production of MIP-2 but not MCP-1 in vitro. Furthermore, this epithelial cell production of MIP-2 required ERK activation that was dependent on TNF-α signaling through TNF-α receptor II. Finally, we adoptively transferred HA-specific TM CD8+ T cells into HA transgenic mice and found that TM CD8+ T cells failed to induce alveolar epithelial cell MIP-2 production and neutrophil infiltration, and produced milder lung injury than transferred wild-type CD8+ T cells. Taken together, these findings suggest that proteolytic processing of tmTNF-α to sTNF-α is a critical event regulating CD8+ T cell mediated injury in a transgenic mouse model of influenza infection.
Evaluation of specific antibodies against prM DENV in Mexican endemic area by using a recombinant protein (67.6)
Authors: Ramírez-Hernández Liliana , García-Cordero Julio , Vivanco-Cid Héctor , Bravo-Maceda Laura , Cedillo-Barrón Leticia
Published: January 2023

Dengue virus (DENV) includes four serotypes, all of them are able to cause DF and DHF. Recently it has described that the antibody response to the prM protein of DENV may plays an important role in both protection from and enhancement of disease. At this respect it has reported that prM-antibodies are highly cross-reactive among the four DENV serotypes and, interestingly at high concentrations, this antibodies do not neutralize infection but potently promote Antibody-Dependent Enhancement (ADE). The production of recombinant proteins rises as feasible alternative to evaluate massively the immunological status against different proteins in ELISA conventional assay. The aim of this work is the cloning and production of prM recombinant protein to evaluate the specific antibody response against this protein, in Mexican endemic areas. The recombinant protein was expressed in a bacterial system of E. coli and subsequently purifies and quantified to use as antigen in ELISA indirect assay. Sera from healthy volunteers and sera from Dengue confirmed donors classified as DF or DHF according with the WHO criteria were tested. Our results confirm that an elevated titters of specific antibodies to the prM protein, seems to associate with a secondary infection and DHF development. The production of this recombinant antigen may be useful, cheap and reliable to evaluate massively the immune response in endemic areas.
Detection of RSV F-specific human mucosal IgA antibody titers utilizing a sensitive electrochemiluminescence platform (67.8)
Authors: Patton Kathryn , Zuo Fengrong , Cherukuri Anu
Published: January 2023

It has been previously shown that mucosal immune responses play a significant role in mediating protection against respiratory viral infections. Respiratory Syncytial Virus (RSV) is a persistent respiratory virus which infects humans throughout life and can cause severe lower respiratory tract infections, particularly in the elderly. Establishing key biomarkers of vaccine-induced immune response is essential to the development of a successful vaccine. In this study, thirty healthy human donors separated into two age cohorts were evaluated: Young Adult (20 to 30 yrs, n=10) and Elderly (65 to 85 yrs, n=20). Plasma samples from the 30 donors were evaluated for neutralizing antibody titers against wild-type (wt) RSV A2 using a micro-neutralization assay and results showed an average titer of ~10.5+2.2 log2 in both age cohorts. In an effort to investigate additional biomarkers, we evaluated nasal wash samples from these donors for wt RSV A2 and RSV-F specific IgA antibody titers. We utilized both a conventional ELISA and a novel Electrochemiluminescence technology to detect the antigen-specific IgA titers. Results from these studies identified the Electrochemiluminescence platform as being a highly sensitive and powerful technology with a wide enough dynamic range to discern subtle differences in mucosal IgA antibody titers. Utilizing such an approach to detect critical biomarkers in dilute nasal wash samples should benefit clinical vaccine development.
Dynamic of T regulatory, Th17, and Tc17 cells and their related cytokines during Nocardia brasiliensis infection in BALB/c mice (99.1)
Authors: Rosas-Taraco Adrian , Perez-Liñan Amira , Perez-Rivera Luz , Salinas-Carmona Mario
Published: January 2023

Actinomycetoma caused by Nocardia brasiliensis induces an inflammatory response with multiple microabscesses and granuloma formation with abundant large cells and mononuclear infiltration. Th1 and Th2 lymphocytes response have been evaluated in N. brasiliensis infection. T regulatory, Th17, and Tc17 lymphocytes are being studied to evaluate their role in several infectious diseases, but there are not data in N. brasiliensis infection. In this study, we evaluated the levels of Treg, Th17 and Tc17, as well as their related cytokines during N. brasiliensis infection in BALB/c mice. Methodology: Mice infected with N. brasiliensis in the footpad were sacrificed at 0, 3, 7, 15, 30, 60 days after infection and levels of Treg, Th17 and Tc17 cells in footpad and spleen were measured by flow cytometry, and their related cytokines (IL-6, IL-10, IL-17A, IL-23 and TGF-β) in the homogenized tissue were measured by ELISA. Results: In footpad, Th17/Tc17 lymphocytes were increased at day 3 (P<0.01), while Treg cells at day 7 (P<0.01). In spleen, Treg cells reached its peak at day 15 (P<0.01) meanwhile Tc17/Th17 at day 3 (P<0.05). Anti-inflammatory cytokines were higher, TGF-β at day 7 and 30 (P<0.01), while IL-10 at day 15. IL-17A and IL-23 were increased gradually during the infection (P<0.01). Finally, IL-6 was increased at day 3 (P<0.01). In conclusion, there is an imbalance of Treg, Th17 and Tc17 in the early stage of N. brasiliensis that could favor chronic disease.
Th17 stimulation and IL17A play critical roles in the Staphylococcus aureus iron regulated surface determinant B (IsdB) mediated protection in a disseminated challenge model. (99.11)
Authors: Joshi Amita , Pancari Gregory , Cope Leslie , Bowman Eddie , Cua Dan , McNeely Tessie
Published: January 2023

We have previously shown that IsdB, a conserved protein expressed by Staphylococcus aureus, induces a robust antibody response which correlates with protection in a murine challenge model. Here we looked at IsdB mediated cellular immune response. First the importance of lymphocytes was examined. Using CB-17 SCID mice we noted that lymphocytes were essential in protection against disseminated S. aureus infection. Neither active nor passive IsdB immunization of CB-17 SCID mice conferred protection against a lethal challenge of S. aureus. Adoptive transfer of CD3+ plus B19+ cells conferred protection in CB-17 SCID immunized mice but not controls. Evaluation of lymphocyte subsets indicated that T cells but not B cells conferred protection. CD3+ or CD4+ T cells conferred protection, whereas CD8+ cells were less active. Neither CD19+ B cells nor CD138highB220intCD19lo plasma cells were protective. Supportive of this data, IsdB immunized Jh mice (IgH chain deficient) were protected against lethal challenge, while nude (T cell deficient) mice were not. In vitro assays indicated that isolated IsdB immune splenocytes produced abundant IL-17A, but not IFN-γ after in vitro stimulation with IsdB peptides. These findings suggest a role for Th 17 cells in IsdB mediated defense against invasive S. aureus infection.
Major histocompatibility complex class II gene diversity and tolerance to avian malaria in Hawaiian honeycreepers (Drepanidinae) (99.15)
Authors: Jarvi Susan , Bianchi Kiara , Farias Margaret , Wiegand Kimberly , Skinner Sarah , Asano Ashley , Czerwonka Christopher
Published: January 2023

Susceptibility to avian malaria differs both within and between species of Hawaiian honeycreepers. Due to their critical function in adaptive immunity, proteins encoded by Mhc genes may be key in the development of tolerance to Plasmodium relictum in low elevation populations of `amakihi (Hemignathus virens). To evaluate gene copy number, 263 Mhc class II peptide-binding region clones were sequenced from a captive family of `amakihi. Phylogenetic analysis suggests a minimum of 10 Mhc class II genes with detection of 35 unique alleles. Species comparisons (total 503 clones, eight birds/species) reveal that Mhc diversity is highest in `amakihi [d = 0.085(0.015)] with 98 distinct alleles detected (average 13.5/bird), followed by `apapane (Himatione sanguinea) [d = 0.068(0.012)] with 66 distinct alleles detected (average 11/bird) and in a highly susceptible species, the i`iwi (Vestiaria coccinea) diversity appears lowest with d = 0.057(0.011) and 66 distinct alleles detected (average 10.6/bird). Within `amakihi, Mhc sequences from birds from low elevation (56 distinct alleles, average 14.25 alleles/bird) and high elevation (50 distinct alleles, average 12.75 alleles/bird) subpopulations appear distinct with detection of only one shared allele. Microarray-based population level analysis (~1,000 individuals) of Mhc diversity in `amakihi detected at least one allele with a significantly higher frequency in low elevation populations than mid or high elevation subpopulations.
Route of Francisella tularensis infection shapes the subsequent immune response (99.2)
Authors: Barrigan Lydia , Steele Shaun , Tuladhar Shraddha , Woolard Matthew , Kawula Thomas , Frelinger Jeffrey
Published: January 2023

The bacterium Francisella tularensis is the causative agent of tularemia. F. tularensis induces a host response that is dependent on the route of infection. Intranasal (i.n.) inoculations are more virulent and require fewer bacteria to produce a lethal infection than intradermal (i.d.) inoculations (10^3 organisms i.n. versus 10^6 i.d.). Interestingly, one day post infection, the bacterial loads are similar in the spleen and lung regardless of the route of infection. We also found that i.d. inoculation resulted in IFN-γ+ T cells in the lung whereas i.n. inoculation produced very few IFN-γ+ T cells and instead many IL-17+ T cells in the lung. Due to the similar bacterial loads systemically after 1 day, but very different host responses, we hypothesize that the adaptive immune response is influenced by local events at the site of infection immediately following inoculation. To test this hypothesis, we infected C57Bl/6 mice i.n. or i.d. and identified cytokine production in the lung and tail at four hours post-infection. Additionally, we have identified alveolar macrophages as the primary cell type infected four hours post-infection, to the exclusion of other myeloid cells and lung parenchyma. Furthermore, we have identified cytokines produced by alveolar macrophages when cultured ex vivo and infected with F. tularensis. These data will help us further understand how the adaptive immune response to F. tularensis is shaped early after infection.
Identification of a putative IgM memory B cell population during bacterial infection (99.31)
Authors: Yates Jennifer , Racine Rachael , McLaughlin Maura , Wittmer Susan , Winslow Gary
Published: January 2023

Humoral immunity and B cell memory are essential components of the adaptive immune response. These elements of immunity have remained largely unexplored in intracellular bacterial infections. Ehrlichia muris is an obligate intracellular bacterium that generates a chronic infection in immunocompetent mice. Chronic infection with E. muris is characterized by long-term IgM production that confers antibody-mediated protection against virulent ehrlichial challenge. We have identified a novel CD19+ B cell population in the spleens of chronically infected mice based on the expression of the cell surface markers CD11c, CD73, and PD-L2. As determined by fluorescence microscopy, these cells are localized in germinal centers of the spleen. Upon stimulation with LPS in vitro, the CD11c+/CD73+/PD-L2+ B cells proliferated and produced antigen specific IgM. BrdU incorporation studies revealed that the population is largely quiescent, and new cells were not recruited to this population during chronic infection. We propose that the CD11c+/CD73+/PD-L2+ B cells we have identified during chronic infection, are long-lived effector/memory cells responsible for the maintenance of long-term immunity during chronic ehrlichial infection.
Immunodominance of the Sm-p40 antigen contributes to the development of severe schistosomiasis mansoni (99.5)
Authors: Finger Eduardo , Finardi de Souza Alessandra , Galante Coimbra Thaissa , dos Santos Daniel
Published: January 2023

In murine schistosomiasis, H-2k CBA and C3H mice develop severe CD4 T cell(CD4) mediated liver pathology(LP) and a high Th1/17 response very focused on a single epitope of the parasite egg antigen Sm-p40(p234-246). Low pathology C57BL/6 mice do not recognize this antigen. A possible reason for this result is that p234-246 exacerbated dominance(ED) polarizes the anti-egg response causing severe LP. To test this hypothesis we: a)analyze the LP of SJL mice whose H-2 haplotype also exhibit ED of a single Sm-p40 epitope and b) analyze the LP of CBA mice whose p234-246 ED was neutralized by an epitope cocktail. Methodology:H-2s Sm-p40 restriction pattern was determined with the rankpep software. SJL LP was documented by comparative liver morphometric analysis(LMA) of infected CBA, C57BL/6 and SJL mice euthanized 7 weeks-post-infection(wpi). The effect of p234-246 ED neutralization was studied by LMA of 3 groups of infected CBA mice euthanized at 7wpi. The 1st group was treated with an emulsion of adjuvant and 4 synthetic epitopes designed to compete for I-Ak with an affinity equal or higher than p234-246, the 2nd was treated with adjuvant alone and the 3rd was not treated. Results:LMA shows that SJL mice develop as severe a LP as CBA mice and that p234-246 ED neutralization prevents development of severe LP. Conclusion:These results show that ED may be pathogenic in CD4 mediated diseases and that its neutralization may be an effective therapeutic strategy that deserves exploration.
Ig G and IgM opsonizing antibodies affect Il-12 and TNF-α cytokine production in macrophages infected with a Nocardia brasiliensis recombinant strain. (99.6)
Authors: Salinas-Carmona Mario , Rocha-Pizaña María , Pérez-Rivera Luz
Published: January 2023

Nocardia brasiliensis is is an intracellular microbe that replicates in host macrophages. We developed an actinomycetoma model in BALB/c mice and showed that IgM but not IgG anti-N. brasiliensis antibodies conferred protection and prevent ed actinomycetoma lesion. The survival or death of the bacterium, once in macrophages, possibly depends on the antibodies used to opsonize N. brasiliensis. We constructed a recombinant strain of N. brasiliensis that produces a green fluorescent protein (GFP) to evaluate virulence and role of IgG and IgM antibodies in cytokine production by macrophages in BALB/c mice. The recombinant N. brasiliensis strain expressing the green fluorescent protein constructed by adding a plasmidic vector containing the GFP gene was used to infect BALB/c mice and macrophages to evaluate phagocytosis and cytokine production with opsonizing IgG and IgM antibodies. N. brasiliensis GFP induces actinomycetoma as well as N. brasiliensis WT. The production of IL-12 with opsonized bacteria increased with IgG and decreased with IgM antibodies. TNF- α production was greater at 24 and 48 hours in non-opsonized bacteria. Opsonized bacteria with IgG and IgM antibodies decreased TNFα production but only IgM isotype antibodies decreased IL-12 production by macrophages. The modified N. brasiliensis strain conserves its property to induce disease.
Human sepsis induces a novel cell exhaustion phenotype in the lung characterized by T cell expression of PD-1 & BTLA and HVEM expression on airway epithelium (99.9)
Authors: Boomer Jonathan , To Kathleen , Bricker Traci , Deppong Christine , Chang Katherine , Walton Andrew , Osborne Dale , Crockford Seth , Swanson Paul , Hotchkiss Richard , Green Jonathan
Published: January 2023

Approximately 200,000 people die annually in the US from sepsis. Sepsis is characterized by an acute “cytokine storm” followed by an immune suppressed state. While apoptosis of innate and adaptive immune cells has been demonstrated, the mechanism for immunosuppression in sepsis is poorly defined. In this study, we analyzed the function and phenotype of cells isolated from lung and spleen of septic patients collected within 120 minutes of death and compared these to cells similarly isolated from non-septic patients. Post-stimulation, splenocytes from septic patients had dramatically reduced secretion of the cytokines IFNγ, TNFα, IL-6 and IL-10. Expression of activating and inhibitory receptors was examined by flow cytometry. Splenic T cells had decreased expression of the IL-7 receptor and elevated expression of CD69. Both lung and splenic T cells had increased expression of the inhibitory receptor PD-1 as well as increased expression of its ligands PDL1 and PDL2 on dendritic cells. BTLA but not CTLA4 expression was detected on the majority of lung T cells from septic and control patients. Interestingly HVEM, the ligand for BTLA, was detected by immunohistochemistry on airway epithelial cells of lungs from septic but not control patients, providing a receptor:ligand interaction with the potential to mediate local immune suppression. These data are consistent with a state of immune cell exhaustion, and provide a novel mechanism for local immunosuppression in the lung.
Interferon-mediated inhibition of T cell proliferation contributes to virus-induced transient immune suppression (105.1)
Authors: Urban Stina , Marshall Heather , Welsh Raymond
Published: January 2023

Vaccine-induced memory is required for protective immunity to pathogens, but many viruses induce a transient state of immune suppression that may contribute to the reduction of vaccine efficacy. We demonstrate here that the production of type I interferon (IFN) during an acute virus infection contributes to this transient immune suppression. Exposure of T cells to IFN-inducers at the same time as antigen had stimulatory effects, whereas pre-exposure to IFN-inducers was inhibitory, indicating that the timing of type I IFN exposure was of essence. Suppression of transgenic T cell proliferation was validated in two models of virus infection, where CD8 T cell proliferation in response to cognate antigen was inhibited 3-9 days after virus infection. Pre-exposure of T cells to the type I IFN inducer and TLR agonist poly(I:C) also had inhibitory effects on CD8 T cell proliferation, as shown by competition between poly(I:C)-pre-treated and control-treated P14 CD8 T cells during a LCMV infection. Poly(I:C)-induced inhibition of CD8 T cell proliferation was transient. T cells lacking type I IFN receptors were resistant to the suppressive effects of poly(I:C), indicating a direct involvement of IFN. Detection of IFN receptor and phosphorylated STAT molecules was reduced in poly(I:C)-pre-treated naive CD8 T cells. Inhibition of CD8 T cell proliferation by exposure to type I IFN prior to antigen stimulation may thus contribute to virus-induced immune suppression.
Pulmonary immunopathology mediated by proteolytic processing of TNF-α by antiviral CD8+ T cells (105.11)
Authors: DeBerge Matthew , Enelow Richard
Published: January 2023

Infection with influenza virus evokes a potent CD8+ T cell response that may contribute to lung immunopathology. Recognition of alveolar anitgen by CD8+ T cells results in significant injury that is critically dependent on TNF-α expressed by CD8+ T cells and is largely mediated by the secretion of chemokines by alveolar epithelial cells and the subsequent pulmonary infiltration. TNF-α is expressed as a transmembrane (tm) protein that is proteolytically processed by TNF-α converting enzyme (TACE) to produce soluble (s) TNF-α. To determine the roles of tmTNF-α and sTNF-α in CD8+ T cell mediated lung injury, we generated CD8+ T cells that express either a noncleavable tmTNF-α or have a deletion in the catalytic domain of TACE. Both T cells are impaired in sTNF-α production but exhibited no defect in other effector functions. In vitro analysis indicated that proteolytic processing of tmTNF-α to sTNF-α was required to induce alveolar epithelial cell expression of macrophage inflammatory protein-2. Furthermore, inhibition of proteolytic processing of TNF-α resulted in reduced induction of alveolar epithelial cell chemokines, less neutrophil infiltration and milder lung injury, though these T cells exhibited no defect in antiviral activity in vivo. This indicates that proteolytic processing of tmTNF-α to sTNF-α is a critical event in the initiation of CD8+ T cell mediated lung injury and represents a potential mechanism of regulation of immunopathology during an influenza infection.
Increased susceptibility to CD8 T cell mediated killing limits the HIV-1 reservoir in naïve CD4 T cells from elite controllers (105.13)
Authors: Leng Jin , Buzon Maria , Yang Yue , Seiss Katie , Pereyra Florencia , Yu Xu G. , Lichterfeld Mathias
Published: January 2023

Elite controllers are HIV-1 infected persons with undetectable levels of HIV-1 replication in the absence of antiretroviral treatment, for reasons that remain unclear. We used a flow cytometric cytotoxicity assay based on the detection of Annexin V in dying target cells to assess the susceptibility of naïve and memory CD4 T cell subsets from elite controllers, HIV progressors, HAART-treated persons and HIV-1 negative individuals to killing by identical HIV-1-specific CD8 T cells. We found that naïve CD4 T cells from elite controllers showed significantly higher susceptibilities to HIV-1-specific CD8 T cell mediated killing than naïve CD4 T cells from all other reference patient cohorts. This increased susceptibility to CD8 T cell-dependent cytotoxicity was not related to higher baseline activation or apoptosis rates. Levels of integrated HIV-1 DNA were significantly decreased in naïve CD4 T cells from elite controllers compared to the alternative patient cohorts, while integrated HIV-1 DNA in memory cell subsets were not different between the study groups. Our data suggest that a higher susceptibility of naïve CD4 T cells to the cytotoxic activities of HIV-1-specific CD8 T cells reduces the HIV-1 reservoir in naïve T cells from elite controllers, and in this way contributes to low cellular reservoirs for HIV-1 in these patients.
RAG-1 (Recombination Activating Gene-1) protein is closely related to herpes virus recombinases: Implications for the origins of the acquired immune system. (105.20)
Authors: Dreyfus David
Published: January 2023

The acquired immune system results from recombination of germ-line DNA V(D)J signal sequences by the RAG-1 recombinase. RAG-1 is a dde magnesium binding recombinase related to both retroviral integrases and transposases. However the closest phylogenetic relative to RAG-1 appears to be a family of recombinases encoded by the herpesviridae termed the DBP (DNA binding proteins) that catalyze strand exchange and possibly other recombination events during herpes virus replication. Both the dde magnesium binding triad and the nonamer binding regions of RAG-1 are present and conserved in the DBP. These observations suggest that initially RAG-1 arose from an ancient insertion of a herpes-like infectious agent into a deuterostome genome prior to the origins of the acquired immune system. The author proposes a model in which the primordial RAG-1 recombinase was selected through its ability to promote viral immune responses through innate immune receptors in ancient deuterostomes prior to the origins of V(D)J recombination.
Bolstering the TG-resident HSV-1-specific CD8+ T cell response via IL-10R blockade (105.26)
Authors: St. Leger Anthony , Hendricks Robert
Published: January 2023

Herpes simplex virus type 1 (HSV-1) infection of mouse corneas leads to establishment of a latent viral infection in sensory neurons of the trigeminal ganglion (TG). Activated CD8+ T cells enter the TG during acute infection and are retained in close apposition to neurons during latency. Fifty percent of the CD8+ T cells in the infected TG of C57BL/6 mice recognize an immunodominant epitope on HSV-1 glycoprotein B (gB498-505), while the remaining CD8+ T cells target subdominant epitopes on 11 other viral proteins. Although both populations appear to be stimulated throughout latency, gB498-505-CD8s show increasing functionality (lytic granule release and IFN-γ and TNF-α production) during latency and appear to have a primary role in blocking HSV-1 reactivation from latency. In contrast, TG CD8+ T cells reactive to subdominant epitopes lose functionality during latency. Three weeks of in vivo anti-IL-10R mAb treatment minimally affected the gB498-505-specific TG resident CD8+ T cells, but did dramatically increase in the number of functional CD8+ T cells specific for subdominant epitopes, and improved control of HSV-1 latency in ex vivo TG cultures. We conclude that CD8+ T cells specific for subdominant epitopes are suppressed in the TG by host secretion of IL-10. Our findings suggest the exciting possibility that rescue from IL-10 suppression will expand the functional repertoire of HSV-specific CD8+ T cells and reduce the likelihood of viral reactivation from latency.
HLA targeting efficiency correlates with human T-cell responses magnitude and with mortality from Influenza-A infection (105.30)
Authors: Hertz Tomer , Oshansky Christine , Roddam Philippa , DeVincenzo John , Caniza Miguela , Jojic Nebojsa , Mallal Simon , Phillips Elizabeth , James Ian , Halloran M. , Thomas Paul , Corey Lawrence
Published: January 2023

Experimental and computational evidence suggests that human leukocyte antigens (HLAs) preferentially bind conserved regions of viral proteins, a concept we term “targeting efficiency,” and that this preference can provide improved clearance of infection in several viral systems. One potential advantage of targeting conserved regions of a virus is the higher likelihood of cross-reactive protection from other viral strains, including those newly introduced, such as pH1N1. We hypothesized that individuals that targeted less conserved regions of influenza- A viruses would have weaker T-cell responses following infection with pH1N1. To test this hypothesis, T cell responses to A/H1N1 (2009) were measured from PBMCs obtained from a household cohort study performed during the 2009-2010 influenza season. We found that HLA targeting efficiency scores significantly correlated with IFNγ ELISpot responses (p=0.042, multiple regression). A further population-based analysis found that the carriage frequencies of the alleles with the lowest targeting efficiencies, A*24, were associated with pH1N1 mortality (r=0.37, p=0.031). Interestingly, these are common in certain indigenous populations in which increased pH1N1 morbidity has been reported. The computational tools utilized in this study may be useful predictors of potential morbidity and identify immunologic differences of new variant influenza strains more accurately than evolutionary sequence comparisons.
Modulation of Anti-Viral and Anti-Bacterial B Cell Responses in Super-infections (105.36)
Authors: Wolf Amaya , Strauman Maura , Mozdzanowska Krystyna , Williams Katie , Shen Hao , Weiser Jeffrey , Erikson Jan
Published: January 2023

Respiratory tract infections (RTIs) with influenza virus are a major global health concern, and together with secondary bacterial infections can cause fatal pneumonia. As B cells contribute to protection through the production of antibodies (Abs), we investigated how concomitant viral-bacterial super-infections alter the generation and maintenance of protective B cell responses in RTIs. We established two models of co-infections with influenza A virus (IAV) and Streptococcus pneumoniae (Sp). The first reflects individuals with a viral RTI that go on to contract a subsequent bacterial infection; the second considers individuals that are asymptomatic carriers of bacteria prior to contracting a viral RTI. Mice infected with Sp following IAV exhibit increased signs of morbidity, whereas mice carrying Sp prior to IAV exhibit less morbidity. Super-infection with Sp following -but not prior to- IAV significantly increases anti-viral serum Abs. Furthermore, the kinetics of the germinal center response and the ASC response in the lung-draining mediastinal lymph node and in the lungs are altered. We demonstrate that anti-viral Ab-specificities and -isotypes are differentially affected. Interestingly, the Ab response to Sp is not impacted in either scenario, suggesting distinct regulatory mechanisms for anti-viral and -bacterial B cell responses in these co-infection models.
Programmed Death-Ligand 1 (PD-L1) expressed on Herpes Simplex Virus Type 1 (HSV-1) infected neurons limits survival of CD8 T cells specific for subdominant epitopes. (105.45)
Authors: Jeon Sohyun , St. Leger Anthony , Cherpes Thomas , Sheridan Brian , Hendricks Robert
Published: January 2023

CD8 T cells that reside in the trigeminal ganglia (TG) of HSV-1 latently infected mice maintain viral latency through persistent interaction with infected neurons. Interestingly, CD8 T cells in TG of C57BL/6 mice exhibit a 1:1 ratio of cells recognizing subdominant versus an immunodominant (gB498-505) HSV-1 epitope. We investigated the role of Programmed Death-1 (PD-1) and its ligand, PD-L1 (B7-H1) in determining this ratio. We found that subdominant CD8 T cells in the TG 1) preferentially express PD-1, 2) are functionally compromised, and 3) proliferate and undergo apoptosis at a higher rate than gB498-505-specific CD8 T cells. Moreover, PD-L1 is preferentially expressed on neurons with a high latent viral load, and its expression is regulated by interferon-γ. In B7-H1-/- mice and bone marrow chimeras lacking PD-L1 only on parenchymal cells, the ratio of subdominant cells to gB498-505-specific cells was dramatically increased (4:1). This was due to increased survival of PD-1+ subdominant CD8 T cells that remained non-functional, and did not augment CD8 T cell protection from HSV-1 reactivation in ex vivo TG cultures. We conclude that PD-1/PD-L1 interaction regulates the survival, but not the function of CD8 T cells specific for subdominant HSV-1 epitopes in latently infected TG. Blocking this interaction alone is not sufficient to reduce HSV-1 reactivation from latency, but might be effective if combined with blockade of other suppressor molecules.
High frequency of lytic EBV-IAV cross-reactive CD8 T cells in EBV sero-negative adults (105.47)
Authors: Watkin Levi , Aslan Nuray , Gil Anna , Luzuriaga Katherine , Selin Liisa
Published: January 2023

The gamma-herpes Epstein-Barr virus (EBV) infects ~90% of individuals globally, establishing a life-long infection. The clinical presentation of EBV infection can range from asymptomatic to severe, occasionally fatal, acute infectious mononucleosis (IM). There is also a strong causal relationship between EBV infection and common malignancies, including carcinoma’s, and Burkitt’s lymphoma. However it is unknown why 5-10% of adults remain EBV-sero-negative (EBV-SN), despite the fact the virus infects the vast majority of the population and is actively shed at high titers even during chronic infection. Here, we show that EBV-SN HLA-A2+ adults possess cross-reactive IAV-GIL/EBV-GLC memory CD8 T-cells that show highly unique properties. These IAV-GIL crossreactive cells preferentially expand, and produce cytokines to, EBV antigens with high avidity. Additionally they are capable of lysing EBV-infected targets and show the potential to enter the mucosal epithelial tissue where infection is thought to initiate. They also possess a T-cell receptor (TCR) repertoire that differs by both organization and CDR3 usage from that in EBV-seropositive (EBV-SP) donors. Our results imply that heterologous immunity may protect EBV-SN adults against the establishment of productive EBV infection, and thus be the first demonstration of heterologous immunity between unrelated pathogens in sero-negative human subjects.
Severity of Epstein-Barr Virus (EBV)-induced infectious mononucleosis (IM) correlates with the frequency of crossreactive influenza A virus-M1 and EBV-BMLF-1-specific CD8 T cells (105.48)
Authors: Selin Liisa , Watkin Levi , Luzuriaga Katherine , Aslan Nuray
Published: January 2023

During EBV-associated IM influenza A-specific crossreactive memory T cells are activated and play a role in disease severity. In HLA-A2+ IM patients, influenza M158 (IAV-M1)-specific CD8 memory T cell responses cross-reacted with two different EBV lytic epitopes, BMLF-1280 (17/29) and BRLF-1190 (19/20). Furthermore, 11/22 IM patients demonstrated some intra-viral cross-reactivity between EBV-BRLF1 and -BMLF1 responses. Disease severity of IM did not correlate with viral load, but instead directly correlated with significantly increased frequencies of crossreactive IAV-M1/EBV-BMLF-1-specific CD8 cells, as well as IAV-M1, EBV-BMLF-1 specific CD8 cells, but not BRLF1 responses. When severity of IM was scored and patients were assigned to either mild or severe groups, disease severity correlated with specific TCR Vb usage in all three tetramer-positive populations suggesting that TcR selection is driving disease outcome. Consistent with IAV-M1 and EBV-BMLF1 responses driving increased immunopathology was the observation that patients with severe disease had significantly more IAV-M1 and EBV-BMLF1 cells producing IFNg/MIP1-b in response to antigen as compared to patients with mild disease. These results suggest that T cell crossreactivity can impact T cell selection and function and ultimately disease outcome. Insights on these issues are important for the intelligent design of vaccines and to develop effect therapeutic interventions for virally induced disease.