bluuebunny/crossref_metadata_2025 huggingface dataset:
CD19 cytoplasmic tyrosines 482 and 513 mediating binding to PI3K are sufficient to reconstitute the T-dependent humoral immune response (IRM8P.713)
Authors: Justement Louis , King R.
Published: January 2023

CD19 is a co-stimulatory receptor on B cells that has been shown to play a critical role in regulating B cell differentiation and the humoral immune response. In mice lacking CD19 expression, B1 and marginal zone (MZ) B cell subpopulations are absent and the T-dependent humoral response is severely impaired in association with defective GC B cell generation and GC responses resulting in loss of class switching, affinity maturation and ultimately the failure to generate Ag-specific memory B cells and Ab secreting cells (ASC) that produce class switched, high affinity Abs. Previous studies have demonstrated the selective mutation of tyrosines 482 and 513 (4/5F mice) in the cytoplasmic domain of CD19 is sufficient to recapitulate the phenotype associated with complete loss of CD19 expression. In the present study, mice were generated in which CD19 is expressed containing only Y482 and Y513 in the absence of the other seven cytoplasmic tyrosine residues (4/5Y mice). Analysis of 4/5Y mice revealed that these residues alone are sufficient to restore B1 and MZ B cell development as well as the T-dependent humoral immune response. Studies to examine mice expressing CD19 with a selective mutation to either tyrosine 482 or 513 revealed a preferential requirement for tyrosine 482 to promote a normal humoral response. These data support the conclusion that CD19 predominantly contributes to normal B cell development and the T-dependent humoral response through its recruitment of PI3K.
Regulation of HSH2 adaptor protein turnover via a novel NF-κB-dependent mechanism is involved in determining the nature of the class switched antibody response (IRM8P.715)
Authors: VanValkenburg Arthur , King R. , Justement Louis
Published: January 2023

The Hematopoietic Src Homology 2 (HSH2) adaptor protein is differentially expressed in mature, peripheral B cell subpopulations; exhibiting high-level expression in B1 and marginal zone B cells versus low-level expression in germinal center B cells, independent of gene transcription. HSH2 plays an important role in regulating the production of class switched antibodies through its effect on B cell class switching and terminal differentiation. Mouse models revealed that over expression of HSH2 in the B cell lineage attenuates the class switched antibody response, whereas reduced expression accelerates the class switched response. Thus, it is of significant interest to delineate the molecular mechanisms by which expression of HSH2 is controlled. Upregulation of HSH2 expression in response to TNF receptor family and TLR agonists occurs in an NF-κB-dependent manner, yet does not involve increased transcription of the HSH2 locus based on analysis of HSH2 mRNA levels. Additionally, maintenance of HSH2 expression is dependent on NF-κB signaling. Inhibition of NF-κB results in the rapid down regulation of HSH2 with a half-life of 1 hour, which is in contrast to the results obtained following treatment with cycloheximide or actinomycin D demonstrating that mRNA for HSH2 is stable and the protein itself has a half-life greater than 12 hours. These data support the conclusion that HSH2 protein stability is regulated post-transcriptionally via a novel NF-κB-dependent mechanism.
Disease stage-specific profiling of miRNA in experimental autoimmune encephalomyelitis (IRM9P.732)
Authors: Singh Jaspreet , Deshpande Mandar , Suhail Hamid , Giri Shailendra
Published: January 2023

MicroRNAs (miRNAs) are small non-coding RNA molecules acting as post-transcriptional regulators of many physiological processes. Their dysregulation is implicated in various diseases, including multiple sclerosis (MS). Experimental autoimmune encephalomyelitis (EAE) is a well accepted mouse model of human MS disease. Many of the discoveries of human MS disease mechanism and drug development have come from studies conducted in EAE model. Depending on the mice background, EAE mice mimic two most common form of human MS; chronic (in C57B/6 mice) and relapsing-remitting (RR, in SJL mice). Dysregulated miRNA levels in biological fluids, such as plasma and urine, represent a new source of biomarkers in MS and EAE. In this study, we report the first systematic identification of disease stage-specific (pre-onset, onset, peak and resolution) miRNA expression in the plasma and urine exosomes of chronic mouse model of EAE. Each disease stage was represented by a specific subset of miRNA expression. We report for the first time identification of miR-155 in SC and urine exosomes of EAE mice much earlier (day 6 post immunization) than disease onset (day 12). Further, we evaluated the drug response of copaxone and metformin on miRNA expression. Identification of disease-stage-specific miRNA biomarkers in non-invasive fluids like urine could be helpful for disease prognosis and for discrimination of clinical subtype, thereby aiding therapeutic decisions and monitoring of therapeutic effects.
Atg7 suppresses inflammatory responses in Klebsiella pneumoniae-infected mice by regulating the p38/IκB/NF-κB circuit (IRM9P.720)
Authors: YE Yan , Wu Min
Published: January 2023

Klebsiella pneumoniae (Kp) is a Gram-negative bacterium that can cause serious infections in humans, particularly invading lungs and intestines. Autophagy-related gene 7 (Atg7) has been shown to be associated with certain bacterial infections; however, the role of Atg7 in host immunity against Kp infection has not been elucidated. Here we showed that Atg7 expression was significantly increased in murine alveolar macrophage (MH-S) cells upon Kp infection, whereas knocking down Atg7 with siRNA increased bacterial burdens in MH-S cells. In addition, using biochemical assays and whole animal imaging analysis, we found that compared to WT mice atg7 knockout (KO) mice exhibited increased susceptibility to Kp infection, with decreased survival rates, increased bacterial burdens, and enhanced lung injury. Moreover, atg7 KO mice induced proinflammatory cytokines and superoxide production in Kp-infected lungs. Similarly, Atg7 silencing in MH-S cells markedly increased levels of cytokines. Mechanistically, we observed that Atg7 depletion regulated p38 phosphorylation which changed IκB binding and inhibited NF-κB nuclear translocation, thus exacerbating NF-κB-mediated cytokine secretion. Collectively, these findings indicate that Atg7 offers resistance to Kp infection by modulating both systemic and local production of proinflammatory cytokines via a p38IκB/NF-κB pathway.
ER stress-induced inflammasome activation promotes liver steatosis (IRM9P.723)
Authors: Zhang Jinyu , Li Zihai , Guo Beichu
Published: January 2023

Recent evidences suggest that obese animal exhibits increased endoplasmic reticulum (ER) stress and inflammation in the liver tissue. However, it is largely unknown how ER stress contributes to hepatic steatosis. In this study, we demonstrated that ER stress induced inflammasome activation and IL-1 beta production, leading to hepatic inflammation and steatosis. Our results show that tunicamycin (TM), an ER stress inducer, caused NLRP3 inflammasome activation, leading to increased IL-1 beta processing and production in macrophages and hepatocytes. We found that caspase-1 knockout (KO) mice treated with TM exhibited a less characterization of hepatic steatosis and inflammation compared with WT mice. Our data also demonstrated that ER stress-induced inflammasome activation and hepatic steatosis were partially attenuated by treatment with ER stress inhibitor tauroursodeoxycholic acid (TUDCA) and IRE1α inhibitor (4µ8C). Notably, hepatocytes from WT mice showed marked hepatocyte pyroptotic cell death with more than a fivefold increase compared with caspase-1 KO mice. Furthermore, our results show that ER stress promoted inflammasome activation and IL-1 beta production through IRE1α pathways. Taken together, our findings demonstrate that ER stress-mediated inflammasome activation plays an important role in liver inflammation and steatosis. Thus, ER stress could be potential targets for a novel therapeutic approach to prevent impaired inflammatory related liver diseases.
Cdk5 mediated coronin-1a phosphorylation is required for PLC-γ 1 mediated generation of inositol-1,4,5-trisphosphate and calcium mobilization after TCR activation (IRM10P.745)
Authors: Pareek Tej , Letterio John
Published: January 2023

The intracellular molecular signatures leading to T cell homeostasis and activation largely remains elusive. We have recently reported an unprecedented but important role for Cyclin-dependent kinase 5 (Cdk5) in regulating T cell activation and function, expanding the repertoire of nonneuronal activities for this kinase. In this study, we generated lineage restricted deletion of Cdk5 in T cells (Cdk5TKO-mice) to avoid embryonic lethality caused by the germ line deletion of the Cdk5 gene. Our results show a profound defect in Ca2+ mobilization, IL-2 production, and proliferation following TCR stimulation of mature peripheral T cells isolated from Cdk5-TKO mice. This phenotype was associated with a decrease in viability of activated T cells and Cdk5TKO-mice were refractory to induction experimental autoimmune encephalomyelitis (EAE), an observation associated with an increase in caspase-3 activity in the spleen of Cdk5TKO-mice following immunization. Here we show Cdk5 mediated phosphorylation of Coronin 1a at threonine- 418 regulates coronin-1a complex formation with phospholipase C-γ1 (PLC-γ1), which is essential for the generation of inositol-1,4,5-trisphosphate (InsP3) upon TCR stimulation, thereby mediating Ca2+ release from intracellular stores that is essential for T cell activation and survival. These findings support a rationale for pursuit of Cdk5-trageted therapeutic strategies for the prevention and treatment of autoimmune as well as lymphoproliferative disorders.
Defining a novel role for the NF-κB pathway in proximal TCR signaling. (IRM10P.746)
Authors: Watson Crystina , Beg Amer
Published: January 2023

In the accepted model of T cell activation, parallel and independent signal transduction pathways activate the transcription factors NF-κB, NFAT and AP-1 to drive clonal expansion of T cells in response to antigen. Surprisingly, we found that CD8 T cells lacking NF-κB subunits p50 and cRel fail to activate NFAT contributing to their severely impaired in vivo responsiveness. We further demonstrated that attenuated NFAT activation was due to diminished Calcium influx upon TCR engagement, and that this reduction was accomplished via sub-par phosphorylation of PLCγ. The functional relevance of impaired PLCγ activation was demonstrated by rescue of p50-/-cRel-/- T cell proliferation through pharmacological initiation of PKC and Calcium pathways. While no deficiencies in expression of TCR complex proteins were seen, we did note defects in early TCR-induced Lck and Zap70 activation, and reduced expression of IKKβ and IKKγ in p50-/-cRel-/- T cells. Beyond established roles in NF-κB activation, we show that IKKβ /γ are also crucial for TCR-induced Lck, Zap70 and PLCγ activation. These findings define a novel pathway of “retrograde signaling” in T cells whereby NF-κB subunits modulate expression of proteins which are crucial for early TCR-induced signaling. Therefore, robust T cell activation occurs in cells exhibiting baseline activation of the pro-survival NF-κB pathway either through TCR tickling or non-TCR pathways.
IL-21 blocks B cell death from BCR-delivered TLR9 agonists and upregulates both AID and T-bet (LYM6P.773)
Authors: Naradikian Martin , Oropallo Michael , Marshak-Rothstein Ann , Cancro Michael
Published: January 2023

A growing literature implicates the B cell receptor (BCR), Toll-like receptors (TLR), and survival cytokines or costimulation in the etiology of SLE and other humoral autoimmune diseases. Our lab has found that BCR-delivered TLR9 ligands yield a unique post-proliferative cell death that limits responses to DNA-linked antigens. Furthermore, death via this mechanism is rescued by the survival cytokine BLyS. However, here we show that BLyS is not unique in its capacity to rescue this cell death. Indeed, T cell-derived survival signals including CD40L, IL-4, and IL-21 similarly rescue B cells from BCR delivered TLR9 agonists. All of these stimuli also induce AID, which is required for class switch recombination and somatic hypermutation. However, IL-21 uniquely upregulates T-bet in the context of TLR9 stimulation. Further, we show that IL-21 induces antibody secretion to the IgG2b and IgG2c isotypes. As these isotypes have been implicated in humoral autoimmunity, our findings suggest that IL-21 may be involved in the production of DNA autoantibodies.
Examining the local response in the mucosa: how microfold cells act as first responders during a bordetella infection (MPF3P.811)
Authors: Goodfield Laura , Muse Sarah , Harvill Eric
Published: January 2023

During a respiratory tract infection, the first interaction between the pathogen and host occurs at the mucosal interface. Microfold (M) cells, specialized antigen sampling cells associated with the mucosa and recently identified in the respiratory tract (Kim, D.Y, et al., 2011), are considered the gateway of the initial immune response because they are constantly binding and taking up soluble and inert antigens as well as commensals and pathogens. To determine how M cells contribute to the generation of an appropriate immune response against an invading pathogen, we investigated the response of villous M cells to the respiratory pathogen B. bronchiseptica using a co-culture system and in the murine nasal cavity. Since M cells must quickly respond to initiate a mucosal immune response following the binding of an antigen, we measured changes in mRNA encoding the cytokines and chemokines interleukin-4, interleukin-17, macrophage inflammatory protein 1 α, and tumor necrosis factor-α in culture as well as the ability of B. bronchiseptica to adhere to M cells. To directly measure the response of M cells to B. bronchiseptica in vivo, laser capture microdissection was used to isolate M cells, and gene expression changes were compared during different stages of B. bronchiseptica infection. These results will inform our understanding of the initial interactions between Bordetella and host cells, which will impact the development of an appropriate immune response.
Multi-drug resistance efflux pumps confer additional resistance against host innate immune defense via induction of genes for biofilm formation and virulence (MPF3P.803)
Authors: Gupta Goutam , Wren Melinda , Ganguly Kumkum , Pardington Paige
Published: January 2023

Multi-drug resistance, resistance-nodulation-differentiation efflux pumps in gram-negative bacteria are membrane protein complexes that extrude a wide variety of drugs before they can act on their bacterial target. However, they are not merely pumps for physical transport of drugs but their expression level is also up-regulated by the presence of drugs inside the bacteria, which, in turn, facilitates the release of quorum-sensing molecules and subsequent induction of genes responsible for biofilm formation and virulence. It is our hypothesis that efflux mediated biofilm formation and virulence expression offer gram-negative bacteria additional resistance not only against drugs but also against host innate immune defense. In order to test our hypothesis, we performed real-time qPCR studies on Fluidigm platform to monitor the expression of (i) the efflux genes and their regulators, (ii) the genes for synthesis of quorum sensing molecules and their regulators, and (iii) the genes for biofilm formation and virulence. For this, we used Pseudomonas aeruginosa and its efflux and quorum sensing mutants. The gene expression studies were performed for planktonic cells and nascent and mature biofilms in the presence and absence of a drug, ciprofloxacin. We were able to show efflux mediated expression of biofilm and virulence genes, which resulted in the abrogation of host innate immune defense in a cellular assay.
Normoxic induction of HIF-1α in macrophages is required for protection from the fungal pathogen H. capsulatum. (MPF3P.807)
Authors: Fecher Roger , Deepe George
Published: January 2023

Histoplasma capsulatum (Hc) is the most common cause of respiratory infection among endemic mycoses. Macrophages (MΦ) are the primary phagocytic cell that engulfs Hc. Recent data has demonstrated that the transcription factor hypoxia inducible factor 1α (HIF-1α) can be induced by pro-inflammatory cytokines or LPS in normoxia. Because HIF-1α regulates a number of factors that are potentially vital in MΦ responses to Hc, we hypothesized that myeloid HIF-1α exerts a critical role in conferring protection to Hc. Indeed, the absence of myeloid HIF-1α led to significantly higher fungal burden at 7 days post-infection and a 75% reduction in survival following Hc infection. In vitro, the HIF-1α isoform i.1 (HIFi.1) and downstream target genes were transcriptionally induced in Hc-infected bone marrow derived MΦ in normoxia. To determine if HIFi.1 induction occurs in vivo, mice were infected with a Hc strain expressing GFP. At 7 days post infection, GFP+ (infected) and GFP- (uninfected) MΦ were isolated from lungs; surprisingly both GFP+ and GFP- phagocytes exhibited induction of HIFi.1 and its target genes. Transwell assays demonstrated that a soluble mediator released from infected cells was partly responsible for HIF i.1 induction. Genetic deletion of TNFR1 or dectin-1 abrogated the in vitro induction of HIFi.1. We propose that dectin-1 mediated MΦ TNF-α production is responsible, at least in part, for induction of HIFi.1; myeloid HIF-1α is instrumental in the immune response to Hc.
Intricate properties of memory B cell development after oral immunization (MUC4P.836)
Authors: Lycke Nils , Bemark Mats , Komban Rathan , Stensson Anneli
Published: January 2023

Recent debate over the ability to generate memory B cells in the gut immune system prompted us to explore a novel model with transgenic NP-hapten-specific GFP+ B cells and oral immunizations with NP-conjugated to cholera toxin (NP-CT). We found that orally immunized mice developed exceptional long term memory B cell populations that resided in B cell follicles in Peyer’s patches, mesenteric lymph nodes and spleen. A majority of the GFP+ memory cells, defined by CD80, CD73 and PD-L2 expression, were IgM+, but also a significant number were IgA+ and in spleen IgG+ memory B cells were frequent. Upon analysis of the NP binding VH186.2 IgA gene sequences between 20-40 % carried high affinity mutations and VH186.2 IgG (bone marrow) and IgA (intestine and bone marrow) gene sequences were clonally related. A challenge immunization after 1.5 years, either orally or i.p, elicited a strong NP-specific gut lamina propria (LP) IgA and serum IgG response, starting from a calculated frequency of 1 NP-memory / total 10.000 B cells in these mice. Gut LP IgA plasma cells increased after challenge from 0.05% long-lived NP-specific IgA plasma cells to >5% NP-specific IgA plasma cells of all LP IgA plasma cells. The boosted IgA plasma cells carried more mutations and higher affinities (>60%) than long-lived memory B cells, demonstrating unexpectedly effective selection and maturational processes in gut IgA memory responses.
Bacterial surface (S-) layer glycosylation orchestrates oral microbial communities by tuning the host immunity (MUC4P.841)
Authors: Settem Rajendra Prasad , Honma Kiyonobu , Sharma Ashu
Published: January 2023

Human mucosal surfaces begin to get colonized after birth with microbial communities, which co-exist in the host by modulating the host immune responses. In this process, some microbes decorate with unique sugars to mold the host immunity conducive to their survival. Recently we have shown that oral pathogen Tannerella forsythia (Tf) exploits a similar strategy to facilitate its survival in the oral cavity, and subsequently cause the alveolar bone destruction by dampening Th17-dependent neutrophil infiltration. The present study was aimed to assess the role of Tf surface O-glycans in the manipulation of host immunity and test if this might impact the colonization and persistence of other co-habitants, such as Porphyromonas gingivalis (Pg). Periodontitis mouse model was utilized to compare the ability of Tf or its O-glycan deficient mutant ED1 in impacting Pg colonization and induced alveolar bone loss. In comparison to the ED1: Pg infected group, higher alveolar bone loss was observed in the Tf: Pg group. This paralleled with osteoclastic numbers. Elevated numbers of neutrophils observed in the ED1: Pg group over Tf: Pg group, associated with bacterial colonization levels. Our study suggests that Tf tunes host immunity to promote its colonization and of its co-habiting partner P. gingivalis, leading to increased alveolar bone loss and this scenario parallels with clinical settings where the increased periodontitis severity has been observed in patients harboring Pg and Tf.
Colonic goblet cell intrinsic, Myd88 dependent, microbial sensing rapidly regulates antigen delivery to the lamina propria immune system (MUC4P.853)
Authors: Knoop Kathryn , McDonald Keely , McCrate Stephanie , McDole Jeremiah , Newberry Rodney
Published: January 2023

The delivery of antigens across the intestinal epithelium is an early and critical event in homeostatic responses to innocuous antigens and inflammatory responses to potential pathogens. The factors and mechanisms regulating antigen delivery to the lamina propria (LP) immune system are largely unknown. We recently demonstrated when small intestinal goblet cells (GCs) secrete, they form goblet cell-associated antigen passages (GAPs) and deliver luminal antigens to LP dendritic cells (DCs). Here we report in the basal state, GAP formation is driven by acetylcholine (Ach) signaling via the muscarinic ACh receptor 4 (mAChR4) expressed by GCs. In contrast to conventionally housed mice, GAPs were present in the colon of mice with a reduced microbiota or altered microbial sensing. Formation of colonic GAPs in these mice was dependent upon mAChR4 signaling. Moreover, in mice with a reduced microbial load, luminal microbial stimuli inhibited GAP formation within minutes. In vitro studies revealed GC intrinsic sensing of the microbiota rapidly inhibited ACh induced GC secretion, and conditional deletion of Myd88 in GCs in conventionally housed mice resulted in the spontaneous formation of colonic GAPs, and the delivery of luminal antigen to colonic LP DCs. These findings identify a role for microbial sensing by colonic GCs to rapidly regulate antigen delivery to the LP immune system, and preventing delivery of luminal antigens when the luminal microbial load is abundant and complex.
Gestational hypothyroidism in mice makes female offspring more resistant to pneumococcal pneumonia (MUC5P.869)
Authors: Nieto Pamela , Peñaloza Hernán , Salazar Francisco , Kalergis Alexis , Riedel Claudia , Bueno Susan
Published: January 2023

Gestational hypothyroidism affects fetus neurodevelopment and leads to cognitive deficiencies. In rodents, this condition also affects the central nervous system and produces differences in the amount and type of immune cells in spleen. We wanted to study if the offspring gestated under hypothyroidism have alterations in the performance and/or activation state of the immune cells during an infection. To evaluate this, we studied the immune response against the pathogen Streptococcus pneumoniae in female mice gestated in hypothyroid mothers (hypo mice) and in control mothers (untreated mice), which were infected intranasally to cause pneumonia. A survival curve showed that hypo mice were more resistant to the infection. Hypo mice also showed an increase in the IL-17 levels in BALF. Other parameters such as immune cell populations, histopathology, and bacterial load were also evaluated. For the first time, our study addresses whether there is a correlation between gestational hypothyroidism and the functionality of the offspring immune cells during an infection.
Both local antigen and inflammation are required to establish resident memory CD8 T cell populations in the lung parenchyma and airways (MUC5P.866)
Authors: McMaster Sean , Wilson Jarad , Wang Hong , Kohlmeier Jacob
Published: January 2023

Microenvironment cues regulating the establishment and maintenance of a pathogen-specific resident memory T (TRM) cell population in the lung parenchyma and airways are poorly understood. We show that local inflammation is sufficient to “pull” systemic antigen-specific effector T cells into the lung and airways acutely, but these cells are not maintained and do not develop into long-lived TRM. However, administration of both local antigen and inflammation is sufficient to foster the establishment of long-lasting TRM populations in both the lung parenchyma and airways; moreover, local antigen and inflammation is able to compensate for the defect in airway-resident T cell memory (TARM) recruitment in mice infected with influenza virus via the intramuscular route as compared to the intranasal route. We also show that mice administered local antigen and inflammation are more protected upon heterologous challenge as compared to mice receiving either local inflammation or local antigen alone. This observation correlates well with our finding that the airway and lung parenchyma TRM populations are not systemically maintained. Follow-up experiments suggest a hypothesis that the lung TRM population could be the replicative reservoir for the TARM population. Our findings could further the development of cellular-mediated vaccines against respiratory pathogens by defining the parameters for the establishment of pathogen-specific TRM populations in the lung parenchyma and airways.
Compensation-free dead cell exclusion: multi-beam excitation of the far-red DNA binding viability dye DRAQ7. (TECH2P.873)
Authors: Edward Roy , Dimmick Ian
Published: January 2023

Reliable exclusion of dead/damaged cells is often needed for immunological assays by flow cytometry and cell sorting. DRAQ7 is a far-red biomarker of cell viability developed for this purpose. This water-soluble molecule binds dsDNA with high affinity but is membrane impermeant, only labeling cells with compromised membranes (Akagi, J. et al., 2013 and Smith, P.J. et al., 2013). Multi-colour analysis is limited by the available spectrum and preference for bright fluors, increasing compensation issues. DRAQ7 aids panel design by its emission: not overlapping Brilliant Violet dyes, FITC or R-PE and minimally with APC. Due to its specificity and affinity it provides clear s:n in membrane-compromised cells. DRAQ7 is optimally excited at 599 / 644 nm, however it is usefully excited down to blue wavelengths. In principle, this, so called, multi-beam excitation means DRAQ7 occupies multiple fluorescence channels. Conversely, this actually enables selection of a unique population of dead cells; plotting two independently excited far-red channels against each other one can choose a suitable pairing that gives the best separation of a double-positive population for a chromophore panel. This double-positive population is defined as a dead-cell gate of events then entirely excluded from other channels (avoiding any compensation). This can be achieved during acquisition or post-acquisition. The technical principles of this simple approach and exemplar data will be presented.
Identification of CXCR2 as an important regulator of granulocytic myeloid-derived suppressor cell mobilization during tumor progression (TUM4P.912)
Authors: Bian Zhen , Shi Lei , Liu Yuan
Published: January 2023

Expansion of myeloid-derived suppressor cells (MDSCs) is associated with cancer progression and suppression of T cell function. However, further defining MDSCs and their role is hindered by the fact that no specific cell marker is available to distinguish MDSCs from non-suppressive leukocytes. Here we report a separation method that clearly separate granulocytic MDSC (G-MDSC) from non-suppressive granulocytes. In this study, mouse bone marrow cells from healthy and tumor bearing mice were collected, followed by Percoll density gradients centrifugation. Naive CD11b+Ly6G+Ly6Clow granulocytes from the fraction between 50-60% Percoll effectively inhibited the proliferation of T cells via production of ROS and arginase. These suppressive cells were expanded during tumor formation and the inhibitory function was enhanced in tumor bearing mice, indicating that G-MDSC was enriched at the 50-60% Percoll interface. Unlike G-MDSC, high density granulocyte (~60-70% Percoll fraction) failed to suppress T cells proliferation but had a high phagocytic activity and chemotatic capacity toward CXCL1, suggesting that they were mature neutrophils. Further study found that CXCR2 in G-MDSC from tumor bearing mice was significantly upregulated, leading to a massive migration of G-MDSC into spleen and tumor site. Collectively, our findings provide a new way to discriminate G-MDSC from mature granulocytes and reveal a critical role of CXCR2 in G-MDSC mobilization during tumor formation.
MDSC-derived and TIL-endogenous NO plays a crucial role in the induction and stability of Th17 cells in human ovarian cancer (TUM4P.915)
Authors: Obermajer Natasa , Wong Jeffrey , Edwards Robert , Chen Kong , Scott Melanie , Khader Shabaana , Kolls Jay , Odunsi Kunle , Billiar Timothy , Kalinski Pawel
Published: January 2023

Intratumoral Th17 numbers and local production of IL-17 have been shown to positively correlate with survival of ovarian cancer (OvCa) patients. We demonstrate that the tumor-associated expression of IL-17A in OvCa patients positively correlates with the expression of NOS-2. Our data indicate that the nitric oxide synthase-2 (NOS2/iNOS)- driven NO produced by patients’ myeloid-derived suppressor cells (MDSCs) promotes the development of RORgt(Rorc)+IL-23R+IL-17+ Th17 cells. In addition to the positive impact of the exogenous (MDSC-produced) NO, we show that the development of Th17 cells from naive-, memory-, or tumor-infiltrating CD4+ T cells, driven by IL-1β/IL-6/IL-23/NO-producing MDSCs or by recombinant cytokines (IL-1β/IL-6/IL-23), is associated with the induction of endogenous NOS2 and NO production, and critically depends on NOS2 activity within CD4+ T cells. Inhibition of NOS2 activity or the canonical cGMP/cGK pathway of NO signaling abolishes the de novo induction of Th17 cells and selectively suppresses IL-17 production by established Th17 cells isolated from OvCa patients. The current results provide rationale for targeting NO, NOS2, cGMP, and cGK as new targets to manipulate Th17 responses in cancer.
Mast cells and mast cell-derived IL-13 play an important role in MDSC activation, migration, and accumulation. (TUM4P.925)
Authors: Damle Sheela , Martin Rebecca , Saleem Sheinei , Folgosa Lauren , Zellner Hannah , Nguyen Kim , Ryan John , Bear Harry , Irani Anne Marie , Conrad Daniel
Published: January 2023

Until recently, the interaction between mast cells (MCs) and CD11b+Gr-1+ myeloid derived suppressor cells (MDSCs) was limited to recruitment of MDSCs to the tumor site. However, we report that MCs are also needed for the activity of MDSCs. Adoptive transfer (AT) of MDSCs failed to promote B16 melanoma colonization in MC deficient mice. MDSCs in these mice also failed to localize to the liver and stayed mainly in peripheral blood. MC sufficient mice accumulated MDSCs in the liver and retained significantly lower levels of MDSCs in circulation after AT. Recently, Ma et al. (Cancer Res. 2013 Jul 1; 73(13);3927-37.) has shown that MC derived IL-13 has been show to promote the growth of pancreatic ductal carcinoma. Given this, we investigated the role that IL-13 plays in the MDSC/MC interaction. Our data shows that IL-13 KO mice crossed to ADAM10Tg mice, which carry a mutation resulting in the over-accumulation of MDSCs, have significantly decreased MDSC accumulation in the naïve state, especially evident in the granulocytic subset. In a model of natural MDSC accumulation (Lewis Lung Carcinoma, i.v.), IL-13 KO mice had reduced tumor colonization to the lungs. They also had reduced MDSC accumulation in the liver and spleen, but increased number in circulation. Taken together, our data indicates that MCs and IL-13, an important MC-derived cytokine, play an important role in MDSC migration and accumulation and represent important drug targets for the control of MDSCs in tumor.
Impact of non-oncogenic acute viral infection on anti-tumor CD8+ T cell responses (TUM5P.938)
Authors: Zloza Andrew , Huelsmann Erica , Lacek Andrew , Lusciks Eugene , Nabatiyan Arman
Published: January 2023

Background: Factors dictating clinical emergence of cancer are not fully understood. Specifically, how non-oncogenic acute viral infections affect unrelated anti-tumor CD8+ T cell responses (those in uninfected tissues) is unknown. Methods: Therefore, we infected B6 mice with non-lethal Influenza (A/PR/8-OVA, 80,000 EID50, intranasally) 3 days prior to challenge with B16-F10 melanoma (120,000 cells, subcutaneously, right flank). To track self/tumor antigen, virus-expressing antigen, and unrelated foreign antigen CD8+ T cells, we transferred pmel (gp100-specific, Thy1.1+Ly5.1-), OT-I (OVA-specific, Thy1.1+Ly5.1+), and P14 (LCMV gp33, Thy1.1-Ly5.1-) CD8+ T cells, respectively, into Thy1.1-Ly5.1+ B6 mice. Results: Influenza infection significantly worsened normal host anti-tumor responses and decreased host survival with cancer from ~32 to ~15 days (P<0.01). Interestingly, pmel (but not OT-I and P14) CD8+ T cells circulating to the infected lung underwent cell death resulting in their eventual loss from the tumor microenvironment (up to 90%; P<0.001). Similar findings were observed with infections 15 or 30 days before or 7 days after B16 challenge and when Balb/c or humanized NSG mice were challenged with breast cancer or melanoma in the context of LCMV or HIV. Conclusions: Our findings may explain the clinical phenomenon emerging from epidemiologic studies reporting increased unrelated cancer in patients with non-oncogenic viral infections.
Developing an antibody targeting CεmX of mIgE for the treatment of allergic and other IgE-mediated diseases (VAC6P.954)
Authors: Chan Yueh-Hsuan , Lee Yu-Tzu , Chou Hung-Wen , Wu Pheidias C , Chen Jiun-Bo , Li Chau-Hong , Cheng Tien-Tien , Chen Nien-Yi , Lue Ko-Haung , Chang Tse-Wen
Published: January 2023

CεmX (also referred to as M1’) is a discrete domain of 52 a.a. residues, located between the CH4 domain and the C-terminal membrane anchor peptide of the ε heavy chain of membrane-bound IgE (mIgE) on human B lymphocytes. Antibodies that target CεmX are potentially useful in controlling IgE production for treating allergic and other IgE-mediated diseases. Based on its abilities to bind to mIgE with high affinity and to lyse mIgE-expressing B lymphocytes by apoptosis, ADCC, and other cytolytic mechanisms, humanized 4B12 monoclonal antibody (h4B12 mAb) is now under preclinical development. In an allergic asthma animal model employing genetically modified mice that express mIgE containing human CεmX domain, we have demonstrated that mAb 4B12 was effective in reducing antigen-specific IgE, but not immunoglobulins of other isotypes. 4B12 could also markedly alleviate airway hyperresponsiveness (AHR) to inhaled methacholine in those mice. Furthermore, we have demonstrated that h4B12 could inhibit the production of human IgE in mice that had been reconstituted with human PBMCs. We have now developed a transfected CHO cell line capable of producing h4B12 at a high yield in preparation for human clinical trials.
Therapeutic clearance of the virally infected nervous system via noncytopathic T cell interactions with resident myeloid cells (VAC6P.945)
Authors: Herz Jasmin , McGavern Dorian
Published: January 2023

The goal of anti-viral therapy is to promote pathogen clearance without causing tissue injury. Therapeutic administration of anti-viral T cells (adoptive immunotherapy) has shown promise in clinical studies for the treatment of CMV, EBV, and adenovirus. Our laboratory models adoptive immunotherapy by transferring anti-viral memory T cells into mice persistently infected from birth with lymphocytic choriomeningitis virus (LCMV). Here we demonstrate that brain-resident myeloid cells (microglia), in addition to neurons, are persistently infected with LCMV in carrier mice. Therapeutic memory T cells can completely purge virus from persistently infected microglia and neurons without causing blood brain barrier breakdown or cellular injury. This is accomplished through a tailored release of chemoattractants that recruit adaptive immune cells, but few pathogenic innate immune cells. In addition, memory T cells enlist the support of nearly all microglia by converting them into CD11c-expressing antigen-presenting cells (APCs). We observed similarly converted myeloid cells in the brains of humans infected with JC virus and HIV-1. Using two-photon microscopy, we revealed that during clearance of the LCMV-infected brain anti-viral CD8 T cells form stable, noncytopathic interactions with CD11c+ microglia, whereas CD4 T cells interact transiently. These data demonstrate that brain-resident APCs can thwart the cytolytic functions of anti-viral T cells while allowing viral clearance.
Use of biologic nanolipoprotein particles containing monophosphoryl lipid A as a novel vaccine platform against inhalational pathogens (VAC7P.956)
Authors: Dunkle Alexis , Blanchette Craig , Boone Tyler , Corzett Michele , Hwang Mona , Fischer Nicholas , Lehmann Doerte , Lychak Cheri , Hoeprich Paul , Driks Adam , Rasley Amy
Published: January 2023

Subunit vaccines are theoretically safe and easy to manufacture but require effective adjuvants and delivery systems to yield protective immunity, particularly at critical mucosal sites such as the lung. We investigated nanolipoprotein particles (NLPs) containing the Toll-like receptor 4 agonist monophosphoryl lipid A (MPLA) as a platform for intranasal vaccination against Bacillus anthracis. Modified lipids enabled attachment of disparate spore and toxin protein antigens. Intranasal vaccination of mice with B. anthracis antigen-MPLA-NLP constructs vs. delivery with free MPLA induced robust IgG and IgA responses in serum and in bronchoalveolar and nasal lavage. Typically, a single dose sufficed to induce sustained antibody titers over time. When multiple immunizations were required, specific antibodies were detected earlier in the boost schedule with MPLA-NLP-mediated delivery than with free MPLA. Administering combinations of constructs induced responses to multiple antigens, indicating potential for a multivalent vaccine preparation. No off-target responses to the species-matched NLP scaffold protein were detected. Using the ovalbumin/TCR transgenic model, MPLA-NLPs enhanced antigen-specific T cell expansion in lung-draining lymph nodes. In summary, the NLP platform enhances cellular, humoral, and mucosal responses to intranasal immunization, indicating promise for NLPs as a flexible, robust vaccine platform against B. anthracis and potentially other inhalational pathogens.
Recombinant outer membrane protein: a potential candidate for Th17 based vaccine against Klebsiella pneumoniae. (VAC7P.967)
Authors: Chen Kong , Goodwin Meagan , McAleer Jeremy , Nguyen Nikki , Way Emily , Kolls Jay
Published: January 2023

Bacterial pneumonia is a leading cause of mortality and one major pathogen associated with this disease is Klebsiella pneumoniae. Recently, the emergence of antibiotic resistant strains demands an effective vaccine against these bacteria. We have previously shown that intranasal immunization with heat-killed K. pneumoniae or crude outer membrane proteins isolated from K. pneumoniae induced antigen specific Th17 responses and these Th17 cells conferred serotype independent protection against various clinical isolates of K. pneumoniae including the recently described multidrug resistant New Delhi Metallo-beta-lactamase-1 strain. To develop a clinically relevant Klebsiella vaccine, we cloned single OMP genes by PCR from K. pneumoniae and successfully expressed one recombinant outer membrane protein, OmpX, in the BL21 E. coli strain. The purified recombinant OmpX was recognized by Klebsiella immune serum by direct ELISA and also recognized by Th17 cells from Klebsiella immunized mice. In vivo, intranasal immunization of the purified OmpX induced robust mucosal Th17 responses and left IL-17 producing gamma-delta T cells unaffected. Ongoing work will determine whether these Th17 responses will result in serotype independent protection against live bacterial challenge including the multidrug resistant strains.
Vaccination with a Hybrid 1 (H1) fusion protein combined with a liposomal adjuvant (CAF01) induced antigen specific T-cells 3 years post vaccination in a human clinical trial. (VAC7P.971)
Authors: Henson Dawn , van Dissel Jaap , Joosten Simone , Graves Andrew , Hoff Søren , Soonawala Darius , Prines Corine , Thierry-Carstensen Birgit , Andreasen Lars , de Visser Adriette , Agger Else , Ottenhoff Tom , Kromann Ingrid , Andersen Peter , Hokey David
Published: January 2023

Approximately one third of the world’s population is infected with Mycobacterium tuberculosis. Developing novel vaccines to protect against pulmonary tuberculosis is a public health priority. In this study, a Hybrid 1 (H1) subunit vaccine containing a recombinant fusion protein of Ag85B and ESAT-6 was paired with a two-component CAF01 liposomal adjuvant system developed and manufactured by Statens Serum Institut. A phase I clinical study was performed to evaluate H1:CAF01 in healthy non-BCG vaccinated adult male and female subjects between the ages of 18 and 55 years old. The subjects were randomized into four groups including H1 alone or H1 with 125/25µg, 313/125µg or 625/125µg CAF01 and were vaccinated on study days 0 and 56. PBMCs harvested approximately 150 weeks post vaccination were used to assess antigen specific responses by a 13-color intracellular cytokine staining assay. Vaccination with H1:CAF01 resulted in statistically significant Ag85B-specific CD4 polyfunctional CD154+ T cells compared to H1 alone. ESAT-6 stimulation resulted in detection of CD4 polyfunctional CD154+ T cells responses that were not elevated to a statistically significant extent compared to H1 alone. This is the first demonstration of the persistence of an antigen-specific cellular immune response up to 3 years after vaccination in a clinical trial using H1:CAF01 vaccination.
CCR10 expression is required for immunogenicity of a HIV-1env DNA vaccine encoding CCL28 to enhance HIV-1env-specific IgG and IgA at relevant mucosal sites (VAC7P.986)
Authors: Kutzler Michele , Kathuria Noshin , Curatola Ashley , Makurumidze Getrude , Myles Devon , Muthumani Kar , Sylvester Albert , Ginsberg Arielle , Hutnick Natalie , Moldoveanu Zina , Hunter Meredith , Khan Amir , Sardesai Niranjan , Mestecky Jiri , Marx Preston , Weiner David
Published: January 2023

Development of a vaccine that drives anti-viral mucosal B cell responses is critical for protection against HIV-1 infection. It is currently unknown whether triggering CCR10/CCL28 pathways in a DNA-based vaccine results in induction of HIV-1env specific B cell immunity at mucosal sites of infection. We hypothesized that co-immunization with HIV-1env/CCL28 molecular adjuvant would augment B cell responses at gastrointestinal and vaginal sites and require CCR10. CCL28 co-immunized WT mice displayed a significant enhancement of HIV-1env specific antibody titers in serum, feces and vaginal washes, and enhanced HIV-1 specific IgA responses were abrogated in CCR10KO mice. CCL28 co-immunization did not increase the breadth of linear B cell epitopes in WT mice, but augmented the dominant epitopes elicited by antigen immunization alone. The frequency of splenic and intestinal IgA+CD19+B220+CD138+CCR10+ plasmablasts was augmented in the CCL28 co-immunized WT mice over antigen-only immunized WT controls. The physiological relevance of these findings was confirmed in a NHP model of intravaginal SIVsmE660 challenge in which CCL28 co-immunization resulted in significant increases in serum/vaginal IgG/IgA, decrease in peak viral loads, significant suppression of viral titers over 120 days, and recovery of CD4 T cells. These data support a role for CCL28 in targeting protective anti-viral B cells to mucosal sites when delivered as molecular adjuvants for HIV-1env DNA-based vaccines.
High-affinity TCRs confer antigen sensitivity in HIV gag-specific effector CD8 T cells (VAC7P.991)
Authors: Wilson Caleph , Robles-Oteiza Camila , Riley James
Published: January 2023

Virus infected cells are eliminated by CTLs when TCR recognizes viral Ag on MHC. Previously, we demonstrated that HIV-1-GAG specific high-affinity (ha) TCR enhanced CD8 T cell killing of HIV-1-infected cells. To elucidate why haTCR expressing T cells were better at controlling HIV-1 replication, we asked whether WT or haTCRs expressing primary human T cells had a distinct cytokine profile when stimulated with monomeric pMHC attached to magnetic beads. In this minimalistic T cell activation system, we observed robust IFNγ, TNFα, IL-2 and MIP1β induction in the presence or absence of CD28 stimulation. Interestingly, αCD3 coated beads do not induce cytokine expression. Previously, we and others demonstrated that αCD3 stimulation failed to induce AKT activation, which has a prominent role in cytokine production. Here, TCR signaling alone activates AKT. Next, we addressed whether T cells expressing haTCRs were able to respond to less Ag, and thereby control HIV-1 replication better by destroying HIV-1 infected cells before they could effectively spread the virus. By mixing various ratios of cognate pMHC with non-cognate pMHC on magnetic beads, we show that CD8 T cells, and not CD4 T cells, expressing haTCRs are able to induce Ag specific activation of phospho-ERK1/2 and -AKT with a ~10 fold increased sensitivity when compared with WT TCR expressing T cells. Thus, implicating CD8 as playing an important role in regulating how a T cell perceives differences in TCR affinity.
Novel vaccination strategy: Francisella tularensis vaccines based on functionalized catanionic vesicles (VAC7P.963)
Authors: Richard Katharina , Mann Barbara , Stocker Lenea , Barry Eileen , Qin Aiping , Cole Leah , Hurley Matthew , Ernst Robert , Michalek Suzanne , Stein Daniel , DeShong Philip , Vogel Stefanie
Published: January 2023

Francisella tularensis (Ft) is a Gram-negative, immune-evasive coccobacillus that causes tularemia for which there is no FDA-approved vaccine. We have utilized a novel vaccine approach using synthetic nanoparticles made from catanionic surfactant vesicles (V), functionalized by incorporation of either Ft type B Live Vaccine Strain (Ft LVS) or Ft type A Schu S4 strain (Ft Schu S4) components (i.e., LVS-V and Schu S4-V, respectively). Immunization of C57BL/6 mice with bare V partially protected against Ft LVS, presumably through activation of an innate immune response, yet failed to protect against Ft Schu S4. In contrast, immunization with LVS-V fully protected mice against intraperitoneal (i.p.) Ft LVS challenge, while immunization of mice with either LVS-V or Schu S4-V partially protected C57BL/6 mice against an intranasal (i.n.) Ft Schu S4 challenge. LVS-V-immunization, but not immunization with V, elicited high levels of IgG against non-LPS epitopes and these antisera conferred passive protection against challenge with Ft LVS. Our recently published and ongoing studies aim to identify the protein targets of mouse antisera, study the mechanism of non-specific protection gained by immunization with this nanoparticle vaccine platform (adjuvant effect) in Ft LVS challenge, and enhance protection in Ft Schu S4 challenge. Our data extend the utility of functionalized catanionic surfactant vesicles as a vaccine platform for pathogens.