bluuebunny/crossref_metadata_2025 huggingface dataset:
Type I interferon negatively regulates acute IL-5 and IL-13 expression in human memory Th2 cells (IRM6P.717)
Authors: Gonzales-van Horn Sarah , Huber Jonathan , Farrar David
Published: January 2023

IFN-α/β is a potent antiviral cytokine that induces the expression of hundreds of interferon-sensitive genes (ISGs). ISGs are positively regulated by IFN-α/β signaling and collectively interfere with various aspects of viral replication. Although the positive regulatory aspects of IFN-α/β signaling through STAT activation have been studied at length, less is known about its role as a negative regulator. Previously, we demonstrated that IFN-α/β suppresses Th2 development in human naïve CD4+ T cells by negatively regulating GATA3 expression, leading to the suppression of Th2 cytokines. In the current study, we found that IFN-α/β also suppressed Th2 cytokine expression from fully committed Th2 cells responding to recall antigen stimulation. Human memory CD4+ T cells acutely stimulated in the presence of IFN-α showed suppressed IL-5 and IL-13 gene expression, however, IL-4 expression was unaffected. Nuclear run-on studies demonstrated that IFN-α signaling suppressed de novo transcription of IL-5 and IL-13, which did not require new protein synthesis. Our data suggest that IFN-α/β was able to acutely repress specific genes by directly inhibiting their transcription. We are assessing direct STAT2 binding by ChIP-Seq, which will identify cis-regulatory regions necessary for gene repression at these cytokine promoters. Taken together, these results demonstrate that IFN-α/β is a key negative regulator of the Th2 response in humans.
The generation of peptide: MHCII-specific TH17 cells in response to intranasal bacterial infection requires IL-6 and TGF-β1, produced from a distinct mucosal dendritic cell subset. (LYM3P.740)
Authors: Linehan Jonathan , Jenkins Marc , Dileepan Thamotharampillai , Cleary Paul
Published: January 2023

Intranasal infections preferentially generate TH17 cells while intravenous infections induce TH1 cells. We explored the basis for these anatomic preferences by tracking endogenous polyclonal CD4+ T cells specific for a p:MHCII ligand derived from the mucosal pathogen Streptococcus pyogenes (Sp). Sp p:MHCII-specific CD4+ T cells were first activated in the cervical lymph nodes (CLN) following intranasal inoculation and then migrated to the spleen and nasal-associated lymphoid organs. The effector cell population generated in the CLN consisted of TH17 cells and TFH cells. Sp-induced TH17 formation was associated with IL-6 and TGF-β1 production by Batf3- CD103- CD301b+ dendritic cells that were located in the CLN but not the spleen. Thus, the tendency of intranasal infection to induce TH17 cells is related to cytokine production by specialized dendritic cells that drain this site.
The coexpression of Tim-3 and PD-1 leads to CD8 T cell exhaustion in aged female mice (LYM4P.760)
Authors: Shin Kwnag-Soo , Lee Kyoo-A , Kim Ga-Young , Kang Chang-Yuil
Published: January 2023

T cells, which are major components of adaptive immune system, are dysfunctional in aged individuals due to numerous intrinsic and extrinsic changes of aged T cell. Recently, several studies have shown that the expression of inhibitory receptors is increased on CD8 T cells from aged mice, which is also observed on exhausted CD8 T cells in chronic infection model, by which effector T cells are negatively regulated. In this study, we described that Tim-3+ PD-1+ CD8 T cells increased in aged mice compared to those of young mice. The more extensive investigations on the functional characteristics of aged Tim-3+ PD-1+ CD8 T cell revealed that these cells had defects in cytokine (IFN-γ, TNF-α) production and proliferation, consistent with features of exhausted CD8 T cell in a chronic infection model. Aged Tim-3+ PD-1+ CD8 T cell produced more immune regulatory cytokine, IL-10, than PD-1 single positive or Tim-3 and PD-1 double negative CD8 T cells. Furthermore, there was more diminished homeostatic proliferation of aged Tim-3+ PD-1+ CD8 T cells than other subsets in lymphopenic conditions, indicating that aged Tim-3+ PD-1+ CD8 T cell had cell-intrinsic defect in proliferation capacity. Interestingly, adoptively transferred aged Tim-3+ PD-1+ CD8 T cells in naïve B6 hosts survived longer than other subsets. Collectively, our findings on aged Tim-3+ PD-1+ CD8 T cell would provide a important insight for understanding the defect in the immune senescence.
Spatial and temporal T cell compartmentalization over the human lifetime: a quantitative, system wide analysis (LYM4P.745)
Authors: Yudanin Naomi , Thome Joseph , Ohmura Yoshiaki , Farber Donna
Published: January 2023

The efficacy of memory T cells in protective immunity has been shown in mouse models, but their lifelong compartmentalization and maintenance in humans remains unclear. To systematically probe lifelong memory T cell generation and maintenance, we have analyzed their distribution and function among 9 tissues acquired through from over 40 healthy donors aged 3 to 73 years old. We statistically analyzed data derived from FACS analysis of these donors, and generated a quantitative map of T cell compartmentalization and maintenance over the human lifespan. Our analyses revealed that differentiated effector memory subsets primarily resided in peripheral tissues while central and naïve subsets remained in circulatory sites. Moreover, expression of the resident memory marker CD69 effectively predicts compartmentalization in peripheral tissues, indicating the need for increased activation to enter the periphery. CD127 and CD28 expression revealed differential maintenance of the subsets in distinct sites, with more cytokine and antigen-driven maintenance prevalent in TEM and TEMRA subsets localized to the periphery. We found that the distribution and maintenance patterns of these subsets are established early in life, and vary little with increasing age. These findings suggest that tissue localization is the primary driver of human T cell maintenance and compartmentalization, and remains stable over the lifetime.
Immunomodulatory effects of IL-10 in tuberculosis granulomas of non-human primates (MPF1P.772)
Authors: Wong Eileen , Gideon Hannah , Flynn JoAnne
Published: January 2023

Tuberculosis (TB), caused by M. tuberculosis (Mtb), continues to be a leading cause of deaths from infectious diseases worldwide. Active TB disease has been associated with suppressed T cell responses and increased production of immunosuppressive cytokines, particularly IL-10. However, published research has focused on the systemic immune response to Mtb as measured in blood. Little is known about the local immune response within individual granulomas. Using a cynomolgus macaque model of Mtb infection, we quantified IL-10 production in granulomas and determined immunomodulatory effects of IL-10 on granuloma immune cells by flow cytometry. Although all granulomas from Mtb infected macaques were found to produce IL-10, the percent of CD3+ T cells that produced this cytokine varied between granulomas, even within an individual host. Neutralization of IL-10 in granulomas ex vivo increased IL-2 and IL-17 production, suggesting IL-10 may normally suppress T cell production of IL-2 and IL-17 in the granuloma. Other cytokines critical for control of TB, such as IFN-γ and TNF, were not affected by IL-10 neutralization. Proliferation was also increased by neutralization of IL-10. Thus, IL-10 may be locally suppressing some, but not all, T cell functions in the granulomas of TB infection. This may be important in either controlling the pathology during infection, or in preventing elimination of the bacilli in granulomas.
Pretreatment of epithelial cells with live streptococcus pneumoniae had no detectable effect on influenza A virus replication in vitro (MPF1P.774)
Authors: Gourapura Renukaradhya , Ouyang Kang , Woodiga Shireen , Dwivedi Varun , Marion Carolyn , Singh Anirudh , Binjawadagi Basavaraj , Hiremath Jagadish , Manickam Cordelia , Schleappi Rose , Khatri Mahesh , King Samantha
Published: January 2023

Influenza A virus (IAV) and Streptococcus pneumoniae (pneumococcus) are two major upper respiratory tract pathogens responsible for exacerbated disease in coinfected individuals. Information on the direct effect of S. pneumoniae on IAV replication in humans is unknown. Such an investigation is important because S. pneumoniae is a commensal of the human upper respiratory tract. The aim of this study was to determine whether treatment of epithelial cells with S. pneumoniae affects IAV replication, determined with the help of a standard immunofluorescence assay (IFA). For this study, four IAV permissive epithelial cell lines including two human-derived cell lines, 12 pneumococcal strains including recent human clinical isolates which represent different genetic backgrounds and serotypes, and six IAV strains of varying genetic nature and pathogenic potential including the pandemic 2009 H1N1 virus were used. Our results suggested that pretreatment of MDCK cells with 7.5×10^6 colony-forming unit (CFU) of live S. pneumoniae resulted in gradual cell-death in a time-dependent manner (1/2 hr to 4 hr). However, pretreatment of cells with 7.5×10^5 and lower CFU of S. pneumoniae had no detectable effect on either the morphology of epithelial cells or on the IAV replication. However, the effect of pneumococci on IAV replication may be different during a coinfection in vivo in the human upper respiratory tract.
Role of IL-17 in chronic pulmonary mycobacterial infection. (MPF1P.775)
Authors: Umemura Masayuki , Fukui Masayuki , Fukui Chiho , Nakae Susumu , Iwakura Yoichiro , Matsuzaki Goro
Published: January 2023

IL-17 family cytokine is comprised of six members, IL-17A to IL-17F. Recent studies using cytokine- and receptor-deficient mice showed that IL-17A and IL-17F were required for responses to extracellular bacterium K. pneumonia in the lungs and C. rodentium in the colon, respectively. However, the involvement of IL-17A and IL-17F in protective immunity was well not clearly demonstrated in mycobacterial infected lung. In this study, we analyzed role of IL-17A and IL-17F in host defense against chronically infected M. tuberculosis. using IL-17A- and IL-17F-KO mice. The IL-17A-KO mice showed significantly decreased survival rates compared with the wild-type (WT) mice during 250-day observation period. In contrast, survival rate of the IL-17F-KO mice were similar to that of the WT mice. Bacterial burdens of various organs of the IL-17F-KO mice on the day 250 were nearly the same as that in the WT mice. In the infected lungs, the IL-17A-KO mice produced less IFN-γ, TNF and IL-6 in comparison to those from the WT mice, while cytokine production of the IL-17F-KO mice were similar to that of the WT mice. This result indicated that the generation of Th1 cells was impaired in the IL-17A-KO mice but not in the IL-17F-KO mice infected with M. tuberculosis. These data strongly support the notion that the lack of IL-17F neither suppress nor enhances protective immunity in the lung after mycobacterial infection.
Role of STAT1 and STAT6 molecules during toxocara canis infection (MPF1P.801)
Authors: Faz Berenice , Ledesma-Soto Yadira , Martínez-Saucedo Diana , Callejas Blanca , Terrazas Luis I.
Published: January 2023

Toxocariasis is a worldwide zoonotic parasitic disease caused by Toxocara canis. Infection is caused by accidental ingestion of embryonated eggs, which hatch and the liberated larvae migrate to different organs. In murine models causes transitory hemorrhagic pulmonary lesions associated with strong TH2 responses and heavy parasite burdens. Alternatively activated macrophages (aaM), through STAT6 signaling, are involved in tissue repair, but its role in T. canis infection has not been determined yet. On the other hand, STAT1 has been clearly related to protection in protozoan infections, but its role on helminths is poorly known. Here we tried to recognize the role of STAT1 and 6 molecules in this infection by using STAT6-/- and STAT1-/- BALB/c mice in determining the outcome of experimental toxocariasis. Mice were infected, and specific antibodies, serum cytokines, parasite burden and lung pathology were analyzed at different times. STAT6+/+ and STAT1-/- mice displayed a Th2 immune response and their lung pathology were less evident compared to STAT6-/- mice, which displayed enhanced Th1 response but more damage in the lung. These differences in lung pathology were associated with the presence of aaM. In the other hand, parasite burden in STAT1-/- and STAT6-/- mice decreased as compared with wild type mice. Our data suggest that the absence of one or another response would contribute to eliminate this parasite, but the presence of aaM is necessary to control tissue damage.
Regulatory B cell induction by Toxoplasma gondii infection implicate in host immune escape and suppression of allergic disease (MPF1P.770)
Authors: Jeong Young-Il , Hong Sung-Hee , Lee Won-Ja , Cho Shin-Hyeong , Lee Sang-Eun
Published: January 2023

Toxoplasma gondii is a worldwide obligate intracellular protozoan parasite that infects humans and animals by either congenital or postnatal routes. Modulation of the immune system by infection with parasites is proposed to suppress the allergic inflammation. In this study we investigated whether experimental infection with T. gondii could induce the immune regulatory cells, including IL-10 producing CD1dhighCD5+ regulatory B cells (Bregs) and CD4+CD25+Foxp3+ regulatory T cells, and whether Breg induction was relevant to parasite immune escape during T. gondii infection. In vivo depletion studies demonstrated an important role for Bregs in T. gondii survival. Furthermore, adoptive transfer of Bregs isolated from T. gondii-infected mice enhanced cyst formation in brain of T. gondii-infected recipient mice. The present study was also investigated whether the development of allergic atopic dermatitis (AD) could be suppressed by T. gondii infection in murine. T. gondii infection substantially suppresses the development AD-like lesions induced by allergen Dermatophagoides farinae in NC/Nga mice, a model of human AD. Collectively, these results indicate that induction of Breg and Treg during T. gondii infection are necessary for evasion of host immunity, and, as the bystander suppressive effect, these cells could suppress allergic inflammation.
Human-derived raw materials: Controlled, consistent collections enable successful manufacturing of cell-based regenerative medicine products (TECH1P.847)
Authors: Ramos Thomas , Wang Wenshi , Okhovat Jacob , McPherson Gaytha , Burger Scott
Published: January 2023

Human cells are critical raw materials for manufacturing cell therapy products, but introduce significant variability. HemaCare controls apheresis procedures and collection sites under a formal quality system, with GMP-compliant, validated procedures and equipment, and GTP-compliant donor screening and tracking. HemaCare performed 86,799 cellular apheresis collections in the last 7 years for research, clinical trials, and commercial products. HemaCare’s unmobilized apheresis products showed consistently high MNC purity, with 93.8% of products containing ≥75% MNC, and an average of 85.66% MNC±7.1% (mean±1 SD). RBC contamination was low, with hematocrit averaging 1.78%±0.7%. Approximately 85% of HemaCare donors have donated apheresis products 5 or more times. HemaCare’s laboratory is equipped with Miltenyi Biotec technology for isolation of raw material into purified cellular subpopulations. The consistency and viability of the purified end products are measured with flow cytometry. Using BioLife Solutions’ serum-free and protein-free cGMP CryoStor™ cryopreservation media with purified cells, post-thaw recovery rates of cell fractions have been above 95%; dendritic cell and macrophage have demonstrated recovery rates of ≥90%. CryoStor™, in combination with freezing in the BioCision CoolCell™ freezing container, has enabled HemaCare to standardize the cryopreservation process, reducing variability while optimizing post-thaw viable cell recovery of its research products.
Automation of immune repertoire amplification on the iC-Processor (TECH1P.851)
Authors: Byrne-Steele Miranda , Pan Wenjing , Brown Brittany , Alleyne Jessica , Yang Qunying , Wang Chunlin , Han Jian
Published: January 2023

Immune repertoire amplification of T and B cell receptors coupled to Next Generation Sequencing (NGS) provides detailed sequence-level insight into the immune system. Previously, we developed a patented, nested multiplex PCR method (arm-PCR) to amplify an immune repertoire, inclusively and semi-quantitatively. Here, we describe the automation of this process in cassette format on the iC-system (iCubate, Inc.) and compare the sequencing results from repeat amplification experiments of the TCRβ and human Ig heavy chains from the same individual. The iC-system is an open, nested multiplex PCR diagnostic development platform, which allows for the development of automated diagnostic panels in a closed cassette format. In addition to the automation, the closed format helps to reduce contamination of the laboratory environment with multiplex primers. Template RNA is added to the cassette at the appropriate concentration with RT enzyme. After two sets of PCR, residual primer-dimers are removed by magnetic selection of the primary product in the cassette. The cassette is then removed from the iC-Processor, and the amplified library is eluted from the magnetic beads. The amplified immune library is directly ready for pooling, QC, and sequencing using the Illumina MiSeq NGS platform. Repeat amplification experiments from the same individual using this automated approach demonstrated high repeatability among shared CDR3 sequences for both the TCRβ and Ig heavy chains.
Predicting conversion to multiple sclerosis using antibody genetics: comparing Sanger and next generation sequencing methods. (TECH1P.858)
Authors: Rounds William , Ligocki Ann , Levin Mikhail , Greenberg Benjamin , Bigwood Douglas , Eastman Eric , Cowell Lindsay , Monson Nancy
Published: January 2023

We have previously identified a distinct mutation pattern in the B cell receptors of cerebrospinal fluid B cells that can predict conversion to multiple sclerosis. This antibody gene signature, developed using Sanger sequencing on singly-sorted B cells, is limited in its clinical application by the time and effort required to obtain such single-cell repertoires. In order to increase the throughput of antibody gene signature evaluation for patients, we used 454 sequencing on bulk-sorted cerebrospinal fluid B cell pellets. We focused our analysis on identifying the mutation and distribution profiles of the repertoires obtained via both sequencing methods. Here we outline the additional filtering and data processing steps used to correct for 454 platform bias and compare the 454 and Sanger sequencing-derived repertoires which include multiple sclerosis samples sequenced by both methods in parallel. Although we observe variable amounts of background mutation and uneven sequence amplification noise by 454 sequencing for our matched samples, most of the antibody gene signature scores remain above the multiple sclerosis conversion threshold established by Sanger sequencing. We expect that additional improvements directed at the sample preparation, data filtering and sequence clustering methods presented here will help further improve the reliability of next generation sequencing-based multiple sclerosis conversion diagnosis.
Over-expression of microRNA-494 is involved in repression of genes but independent of antigen receptor-mediated apoptosis in a T cell leukemia cell line (TECH1P.867)
Authors: Jayaraman Sundararajan
Published: January 2023

Treatment of the human T cell leukemia cell line, Jurkat with the histone deacetylase inhibitor, Trichostatin A (TSA) resulted in histone hyperacetylation, dissipation of mitochondrial transmembrane potential, and augmentation of T cell receptor mediated apoptosis. Global gene expression profiling of TSA-treated Jurkat cells revealed the repression of several genes involved in T cell receptor signaling and PVRIG, a non-coding member of the paired immune-like receptor family genes. Analysis of total RNA by qRT-PCR validated these findings. Genome wide microRNA expression profiling uncovered drug-mediated down-regulation of microRNAs, including the miR-17-92 cluster, critical for T cell biology. Notably, TSA treatment induced the up-regulation of miR-494, which correlated with Jurkat cell death. This is in stark contrast to previous reports indicating that the up-regulation of miR-494 is essential for the survival of carcinomas. Interestingly, transfection of Jurkat cells with a miR-494 inhibitor partially blocked TSA-mediated down-regulation of PVRIG expression, without abrogating the antigen receptor mediated apoptosis. These data demonstrate that the over-expression of miR-494 as a consequence of histone hyperacetylation results in tumor cell death, which is unrelated to antigen receptor-mediated apoptosis. Thus, up-regulation of miR-494 via epigenetic mechanisms appears to be a novel treatment strategy for T cell leukemia.
Mild cold stress resulting from standard housing conditions for laboratory mice influences baseline dendritic cell properties (TUM2P.896)
Authors: Kokolus Kathleen , Spangler Haley , Povinelli Benjamin , Farren Matthew , Lee Kelvin , Repasky Elizabeth
Published: January 2023

The ability of dendritic cells (DC) to stimulate and regulate T cells is critical for effective anti-tumor immunity. Therefore, it is important to recognize any inherent factors which may influence DC function under experimental conditions, especially in laboratory mice which are commonly used to model anti-tumor immunity. Recent data shows that CD8+ T cell frequency within the tumor microenvironment and anti-tumor function is dependent upon the ambient temperature used to house laboratory mice. As DC function is crucial for CD8+ T cell activation, we hypothesized that DC function is also dependent upon housing temperature. We observed increased numbers of splenic DCs (CD11c+ ) in tumor-bearing mice at housed at standard temperature (ST; 22°C) vs. mice housed at a thermoneutral temperature (TT; 30°C) which alleviates the mild cold stress experienced at ST. Despite increased frequency, DCs from mice at ST primarily display immature phenotypes (MHC II+CD86- ), likely rendering them tolerogenic and unable to activate T cells. Further, splenocytes from tumor-bearing mice at ST were unable to induce T cell proliferation following tumor inoculation while DCs from mice at TT elicited T cell activation. These findings likely contribute to faster tumor growth observed in mice at ST vs. TT. This data strongly suggests that the housing temperature can affect fundamental properties of DC function which in turn influence the ability of DCs to regulate the anti-tumor immune response.
IL-33 precedes IL-5 in regulating early commitment to eosinophil development (HEM5P.229)
Authors: Johnston Laura , Berdnikovs Sergejs , Krier-Burris Rebecca , Hsu Chia-Lin , Chien Karen , Bryce Paul
Published: January 2023

IL-33 is strongly associated with allergic disease, in part because of the potent effects it exerts on Th2 lymphocytes, ILC2s, mast cells, and eosinophils, which express its receptor ST2. However, ST2 has also recently been observed on hematopoietic progenitor cells. The effect of IL-33 on these cells is unclear. We observed that naïve ST2KO and IL-33KO mice had significantly less peripheral blood eosinophils (PBEs), whereas exogenous IL-33 was sufficient to elevate PBEs in WT and IL-33KO mice, but not in ST2KO mice. This was associated with increased myeloid/erythroid cell ratios in the bone marrow itself. IL-33 treatment expanded a Sca1-/Linint/SiglecF+ population, characteristic of a committed eosinophil progenitor. Using in vitro eosinophilopoesis conditions, IL-33 promoted mature eosinophil commitment (Sca1-/Lin+/SiglecF+) that was significantly diminished in ST2KO cells. Instead, ST2KO cells accumulated a Sca1-/Lin-/SiglecF+ population, characteristic of an early committed precursor. In contrast, IL-5, which has been shown to drive eosinophil development, expanded mature eosinophils. Finally, we assessed the hierarchy of IL-5 and IL-33 by crossing IL-5 overexpressing transgenic mice (NJ1638) with ST2KO mice. While IL-5 transgenic mice exhibited a robust age-dependent increase in PBEs, ST2 deficiency significantly prevented this IL-5-driven eosinophilia. Our findings demonstrate a novel role for IL-33 and ST2 in supporting eosinophil development that precedes IL-5.
Reduced kinase inhibitory region containing suppressor of cytokine signaling molecule levels in SLE patients is correlated to enhanced STAT1 activation (HUM3P.242)
Authors: Sukka-Ganesh BhagyaLaxmi , Wilson Tenisha , Westley Reeves , Johnson Howard , Larkin Joseph
Published: January 2023

Systemic Lupus erythematosus (SLE) is a chronic inflammatory autoimmune disorder with unknown etiology. Given the importance of the suppressor of cytokine signaling (SOCS) family of intracellular proteins in the regulation of lupus promoting cytokine signaling, we tested the hypothesis that SOCS deficiencies would be present in SLE patients. We therefore compared a group of 34 SLE patients, by disease activity (SLEDIA) and prednisone treatment, to 11 healthy controls for the presence of the kinase inhibitory region (KIR) bearing SOCS1 and SOCS3. We found that protein and mRNA levels of SOCS1 and SOCS3 were significantly reduced in PMBC’s isolated from SLE patients compared to controls. Further, decreased SOCS1, but not SOCS3 protein levels in the SLE patients was inversely correlated to pSTAT1 activation, but not Erk 1/2 or Akt. Notably, the inverted SOCS1/pSTAT1 ratio correlated to significantly enhanced MHC class II levels amongst SLE patients. Together these data suggest that SOCS1 deficiencies may play an essential role in the onset/progression of SLE in a subset of SLE patients, possibly through dysregulated STAT1 mediated antigen presentation. These studies represent a critical first step in implicating a role of SOCS1 and SOCS3 deficiencies in the progression of human SLE. Moreover, these studies point to the possibility that peptides, which partially mimic SOCS signaling and inhibit rodent autoimmune disease, may have efficacy in the treatment of human SLE.
The potential involvement of plasmablast antibodies in clinically isolated syndrome and multiple sclerosis (HUM3P.268)
Authors: Monson Nancy , Rivas Jacqueline , Ligocki Ann , Rounds William , Ireland Sara , Ma Lisha , Frohman Elliot , Greenberg Benjamin , Cowell Lindsay , Stowe Ann
Published: January 2023

Plasmablasts are a transient B cell subtype that secretes large amounts of antibody and arises in response to antigen exposure and T cell help. Once plasmablasts form they die off in a few days or differentiate into a long lived plasma cells, and therefore are only present during an active immune response. In our laboratory a subset of Clinically Isolated Syndrome (CIS) patients presenting with Transverse Myelitis (TM) symptoms at high risk to develop Multiple Sclerosis (MS) also have an elevated level of plasmablasts in the peripheral blood and/or the cerebrospinal fluid. Interestingly, only those patients in our cohort who have elevated plasmablast levels continue to accumulate brain MRI activity. The antibody genetics of plasmablasts from CIS TM patients are altered in comparison to those of plasmablasts responding to influenza from otherwise healthy individuals. There is increased evidence of receptor editing in CIS patients, as well as enrichment of the Antibody Gene Signature (AGS), which predicts whether CIS patients will convert to MS later in life. Antibodies from CIS plasmablasts that are enriched in overall mutations and AGS mutations recognize mouse brain by ELISA and histology, binding to multiple different cell types. By flow cytometry we have shown that many of these antibodies recognize mouse astrocyte and neuron cell lines. If these antibodies are able to bind brain tissue in vivo, they could exacerbate brain damage and contribute to disease in CIS patients.
A second wave of monocyte expansion may contribute to the severe dengue in 2014 outbreak in China (HUM4P.272)
Authors: Yu Lei , Hong Wenxin , Zhao Lingzhai , Zhang Ling , Wang Qianqian , Lei Yang , Qiu Shuang , Yin Chibiao , Feng Hanping , Guan Yongjun , Zhang Fuchun
Published: January 2023

The 2014 dengue outbreak, the largest one in the last ten years in China, resulted in 13,800 hospitalized patients and more than 300 severe dengue cases in Guangzhou. Both of the viral strain and the host immune background may contribute to severe dengue. Here we report the monocyte response in dengue fever (DF) and severe dengue (SD) during the acute infection of seven dengue patients (DF n=4 and SD n=3) using multiparameter flow-cytometry with a panel of 9-surface markers. We also compared dengue specific IgG, IgM and NS1 in serum at the same time points for each patient. Six of the seven patients were diagnosed as secondary DENV1 infection, while one DF diagnosed as primary DENV1 infection. NS1 was nearly cleared in all patients when the serum IgG and IgM reached the high level at day 8-10 from the onset of fever. Obvious monocyte expansion was observed in both of DF and SD groups, frequently companied with the expansion of CD14++CD16+ subset. But the pattern of the response in the two groups was different at the recovery phase. The monocyte expansion in DF group maintained at a high level at day 7-10. Interestingly, the SD group underwent a second wave of monocyte expansion, in parallel with clinical course, starting from day 7 to day 10, especially the CD16+CD64+ monocyte subset. The characteristics of monocyte responses in DF and SD groups will advance our understanding of human dengue pathogenesis and may implicate the potential targets for early therapy.
Severity of infectious mononucleosis correlates with the frequency of crossreactive influenza A/Epstein Barr virus-specific CD8+ T cells (HUM4P.301)
Authors: Selin Liisa , Watkin Levi , Gil Anna , Mishra Rabinarayan , Luzuriaga Katherine , Aslan Nuray
Published: January 2023

During EBV-associated infectious mononucleosis (IM) IAV-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 crossreacted with two different EBV lytic epitopes, BMLF1280 (17/29) and BRLF1109 (19/20). Disease severity of IM directly correlated with significantly increased frequencies of crossreactive IAV-M1/EBV-BMLF1, IAV-M1, and EBV-BMLF1 specific CD8 cells, and with mean viral load over the first 5 weeks of infection. Disease severity did not correlate with BRLF1 or M1/BRLF1 crossreactive 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 IAV-M1 population suggesting that TcR selection is driving disease outcome. Consistent with crossreactive responses driving increased immunopathology was the observation that tetramer-sorted crossreactive responses had completely altered immune response signatures than cognate responses by gene array. These results suggest that T cell crossreactivity impacts T cell selection and function and ultimately disease outcome. Insights on these issues are important for the intelligent design of vaccines and to develop therapeutic interventions for virally induced disease (NIHAI49320).
Basophil surface expressions of CD203c and CD63 following peanut allergen ex vivo stimulation correlate with severity of allergic reactions during food challenge (HYP4P.307)
Authors: Rosa Jaime , Chinthrajah Rebecca , Dominguez Tina , Brock Emily , Nadeau Kari
Published: January 2023

Introduction: Food allergies are a potentially fatal disorder. However, reliable prognostic markers for predicting the severity of allergic reactions during exposures have not yet been established. Recent studies have found that basophil activation tests (BATs) may be a useful adjunct for the diagnoses of peanut and other food allergies. We hypothesize that BAT can also be a useful biomarker for predicting reaction severity during food challenges. Methods: 19 (5-25 years old) peanut-allergic participants (PA) and 5 age-matched healthy controls (AC) who enrolled in our Stanford IRB and FDA approved double blind placebo controlled food challenge (DBPCFC) protocol were given escalating oral doses (5, 20, 50, 100, 100, 100, 125 mg) of peanut and placebo (oat) on separate occasions. The DBPCFC was stopped at the first sign of an objective symptom, which was graded on the modified Bock’s scale. Prior to the DBPCFC, basophils gated via flow cytometry from 200 μl of whole blood obtained from all subjects were stimulated with peanut ex vivo for 20 minutes. Results: For PAs, 8 had grade 1, 7 had grade 2, and 4 had grade 3 reactions to peanut; the ACs had no symptom. Using linear regression, high correlation coefficients were found between Bock’s reaction grades and basophil surface expressions of CD203c (r2 =0.89) and CD63 (r2 =0.85), both p<0.001. Conclusions: Our findings suggest that suitable BATs can be a useful biomarker for predicting clinical reactivity during peanut challenges.
Peanut allergy: a mouse model comparison (HYP4P.309)
Authors: Jhaveri Devi , Bonfield Tracey
Published: January 2023

Peanut induced anaphylaxis often results in a bi-phasic response, associated with a second anaphylaxis once current treatment interventions have been removed. Previous reports have suggested that this response is associated with elevated levels of platelet activating factor. Our studies have focused on refining the murine model of peanut anaphylaxis with the hypothesis that we can generate a model that recapitulates the anaphylactic response to peanuts. We used BALB/c and C3H/HeJ mice (N=20 for each study) mice were sensitized weekly for 6 weeks with crude peanut extract in cholera toxin. Mice were then grouped for crude peanut extract or saline challenge by intra-peritoneal injection. In all mice, temperatures and clinical scores were accessed both early post injection as well as 2 hours later. Serum, lungs, spleen, and liver were stored from all animals for future studies. The BALB/c strain had a mean temperature change from baseline of 0.2 C⁰ for the controls initial post IP versus the 1.89 C⁰ for the challenged mice. The C3H/HeJ strain had a mean temperature change from baseline of 0.1 C⁰ for the controls at the initial post IP versus the 4.2 C⁰ change for the challenged mice. Similar results were noted at the 2 hour mark with the C3H/HeJ strain with the greater temperature change. Both strains had notable changes in clinical scores between the control and challenge groups. This was more pronounced in the C3H/HeJ mice both clinically and by temperature measurement.
Existing allergic inflammation alleviates influenza infection (HYP5P.319)
Authors: Lee Debbie , Prabhu Nayana , Kemeny David
Published: January 2023

Asthma is a disease suffered by millions throughout the world and its prevalence continues to increase. During the 2009 influenza pandemic asthma was found to be the commonest comorbid illness of patients admitted to hospital. It was also observed that asthmatics admitted to hospital with influenza infection were less likely to die or require admission to intensive care compared with non-asthmatics. Using an in vivo model of asthma and influenza infection we demonstrate that prior exposure to Blomia tropicalis extract (BtE) led to a reduction in weight loss and faster recovery following influenza infection compared to PBS/influenza infected mice. This was accompanied by an earlier increase in both CD4 and CD8 T cells in the lungs, which produced more INF-γ, TNF-α and expressed higher levels of CD107a at day 8 post infection. At day 14 post infection levels of T cells in the lungs of the BtE/influenza group were significantly reduced, which parallels the faster recovery from weight loss seen in this group. In summary, we have shown that prior exposure to BtE leads to an altered immune response to influenza infection, comprised of less severe weight loss and the faster recruitment of more highly activated T cells in to the lungs. The earlier recruitment of T cells led to faster resolution of disease and a reduction in T cell numbers. We believe this data provides a clue as to why asthmatic patients present with less severe disease when infected with influenza.
Aging-related changes in lymphatic collectors predispose to pathogen dissemination in tissues (INC4P.342)
Authors: Nizamutdinova Irina , Zolla Valerio , Scharf Brian , Clement Cristina , Maejima Daisuke , Akl Tony , Nagai Takashi , Luciani Paola , Leroux Jean-Christophe , Halin Cornelia , Stukes Sabriya , Tiwari Sangeeta , Casadevall Arturo , Jacobs William , Entemberg David , Condeelis John , Fooksman David , Zawieja David , Gashev Anatoliy , Santambrogio Laura
Published: January 2023

Herein we analyze how the aging process affects the structure and functionality of the lymphatic collectors (LCs) with reference to their ability to maintain pathogen clearance. Ultrastructural, biochemical and proteomic analysis indicated a loss of extracellular matrix proteins, an increase in protein oxidative modifications as well as activation of nuclear factor-κB signaling as sign of “low grade” inflammation in aged LCs. This resulted in a decrease in contractile and pumping activity of LCs, as measured in vivo. Functionally, this impairment also translated into a reduced ability for in vivo bacterial transport as determined by time-lapse microscopy. Ultrastructural and proteomic analysis also indicated a decrease in the thickness of the endothelial cell glycocalyx and loss of gap-junction proteins in aged LCs. Redox proteomic analysis mapped an aging-related increase in the glycation and carboxylation of endothelial cell glycocalyx structural proteins. Functionally, these modifications translated into higher ability of the pathogen to escape from aged LCs into the surrounding tissue. Altogether, our analysis mapped the complexity of the aging-related anatomical, biochemical and functional changes in LCs. The decreased ability to transport bacteria to the draining nodes, associated with increased bacterial escape in the surrounding tissue can contribute to the decreased ability of the immune system to clear pathogens in the elderly, as observed in immunosenescence.
A surprising role for COX-2 signaling in promoting IL-22 in the absence of IL-33 or TLR9 during invasive aspergillosis (INC4P.326)
Authors: Garth Jaleesa , Reeder Kristen , Gessner Melissa , Godwin Matthew , Dunaway Chad
Published: January 2023

IL-33 is a proallergic cytokine thought to play a role in severity of fungal asthma whereas TLR9 signaling has been previously reported to constrain lung immune responses to invasive aspergillosis (IA). We have previously reported a critical role for IL-22 in host defense during IA and therefore questioned whether IL-33 or TLR9 signaling affected IL-22-mediated immune responses. Results show that mice deficient in the receptor for IL-33 (Il1rl1-/- mice) demonstrated enhanced clearance of A. fumigatus from the lungs, which correlated with elevated IL-22 production independent of elevated IL-23 levels. Similar results were observed with mice deficient in TLR9 (Tlr9-/- mice). To this end, we show that PGE2 was induced at higher levels in both Il1rl1-/- and Tlr9-/- mice. In contrast, PGD2 was not detected in either Il1rl1-/- and Tlr9-/- mice whereas PGF2α was observed to be higher in Il1rl1-/-, but not Tlr9-/-, mice. Moreover, treatment of normal mice with IL-33 resulted in attenuated IL-22 induction after A. fumigatus exposure, which correlated with lower PGE2 production. Finally, treating either Il1rl1-/- or Tlr9-/- mice with Celecoxib, a nonsteroidal anti-inflammatory drug which inhibits COX-2 (PGE2/PGF2α induction), attenuated IL-22 production and eliminated the enhanced fungal clearance. These data suggest that signaling via IL1RL-1 and TLR9 regulates COX-2 mediated IL-22 during lung infection with A. fumigatus.
The flagellin D2/D3 domain induces TLR5- and inflammasome- independent antibody responses (INM2P.357)
Authors: LU CHUNCHI , Lopez-Yglesias Americo , CHEN HSINHUI , Zhao Xiaodan , Chao Tiffany , VandenBos Tim , Strong Roland , Smith Kelly
Published: January 2023

The detailed understanding of the molecular mechanisms underlying the biological activity adjuvants is critical for rational design of more effective vaccines. Bacterial flagellin, a potent immunogen, is being developed as a new adjuvant and platform for human vaccines. We have demonstrated that the conserved D0 and D1 domains of flagellin promote antibody responses through activation of their respective innate immune receptors, but are not required for Salmonella flagellin (FliC) induced antibody responses. We hypothesized that additional flagellin domains also influence specific aspects of flagellin-induced B cell responses. Mice were immunized with a mixture of FliC mutants proteins and ovalbumin, and sera were collected for quantification of anti-flagellin and anti-ovalbumin specific antibody responses. The results from our studies demonstrate that the D2/D3 domain of the FliC is also required for flagellin-induced primary antibody responses and robust secondary responses. D2/D3 domain is capable of inducing antibody responses independent of TLR5 and the inflammasome. Our data indicate the flagellin D2/D3 domain synergizes with the TLR5 (D1) and inflammasome (D0) activating domains to induce potent antibody responses, and that the D2/D3 domain also functions as an adjuvant to promote antibody responses against co-administered antigens. Future studies will elucidate the contribution of the FliC D2/D3 domain to B cell responses and its utility in vaccines.
Identification of a novel protein protein interaction pathway with NLRC3 (INM2P.359)
Authors: Davis Beckley , Tocker Aaron , Talento Suzanna , Jacob Kimberly
Published: January 2023

Nucleotide binding domain, leucine rich repeat CARD containing protein 3 (NLRC3) is a member of the NLR gene family. Members of this family have been associated with human inflammatory diseases such as Crohn’s disease and cryopyrin-associated periodic syndrome. Although NLRC3 (and others) are not associated with human diseases, it is expressed preferentially in the immune system and functions in pathogen recognition. NLRC3 is an intracellular protein involved in the sensing of lipopolysaccharide and cytosolic nucleic acids. NLRC3 is hypothesized to act as a negative regulator in response to bacterial and viral infection, suggesting that the vertebrate immune system has evolved specific inhibitors to limit the inflammatory response. We performed an unbiased yeast two-hybrid screen using an amino terminal fragment of NLRC3 to identify putative interacting proteins that might help elucidate the mechanism by which NLRC3 might inhibit inflammatory responses. To this end, we identified several interacting proteins. One protein in particular acts as a scaffold important in regulating the cytoskeleton, cell adhesion and proliferation. Structure function analysis has localized the domains necessary and sufficient for interacting with NLRC3. Additionally, confocal microscopy demonstrates that these two proteins colocalize in transformed human epithelial cells. This data suggests that NLRC3 interacts specifically with a novel scaffold.
Chronic pulmonary exposure of iron oxide nanoparticls induced Th1-type immune response accompanying suppression of antigen-presenting function (IRM12P.647)
Authors: Park Eun-Jung , Lee Sang , Lee Kyuhong , Kim Younghun , Lee Byeong-Seok
Published: January 2023

Accumulated evidence shows that pulmonary nanoparticles induce adverse health effects by modulating immune system in the body. In this study, we investigated local and systemic immune response following a single intratracheal instillation of iron oxide nanoparticles (FeNPs, the oxidized form of magnetite). FeNPs had a needle-like shape, and hydrodynamic area in suspension is 101.3 ± 4.2 nm. At day 90 after a single instillation (0.5, 1, and 2 mg/kg), FeNPs remained in the lung, and particle-laden macrophage increased a dose-dependent manner in BAL fluid. The number of total cells and the portions of neutrophils and lymphocytes significantly increased at 2 mg/kg dose. We also observed that the percentage of apoptotic cells and LDH release increase in a dose-dependent manner. Moreover, Th1-type cytokines were dominantly secreted accompanying the elevated secretion of chemotaxis-related cytokines, including GM-CSF, MCP-1, and MIP-1, in BAL fluid. Distribution of T cells and helper T cells relatively increased along with the enhanced production of IgG and IgM, but not IgE. Additionally, we found that expressions of antigen-presenting-related proteins are suppressed on splenocytes by FeNPs exposure. Taken together, we suggest that chronic pulmonary of FeNPs may locally induce Th1-type immune response, and systemically suppress function of antigen-presenting cells. We also consider the possibility of delayed-type hyperresponsiveness following FeNPs exposure.
Tetanus toxin C fragment immunization generates a robust localized tetanus-binding B cell population (LYM5P.712)
Authors: Kenderes Kevin
Published: January 2023

A long-term objective of our studies is to identify, isolate, and characterize rare antigen-specific B cells. Our approach for identifying B cells has been to use flourophore-conjugated multivalent B cell antigens that can bind to cognate B cells in vivo. We generated a recombinant monobiotinylated tetanus toxin C fragment (TTCF; described in Blood:118:348) and tetramerized it using fluorescently-labeled streptavidin. To generate a population of antigen-specific B cells, we immunized mice subcutaneously with TTCF, in alum; the cells were collected from the draining axial and brachial lymph nodes for flow cytometric analysis. Immunization generated a striking number of TTCF-binding B cells which were detected at frequencies of over 90 percent of the lymph node B cells on day 9 post-immunization; B cell frequency decreased to a few percent of total B cells by day 30. TTCF binding was neither detected in transgenic mice that expressed a hen egg lysozyme-specific BCR, nor in mice immunized with an irrelevant antigen. We propose three hypotheses to explain the extraordinary high frequency of TTCF-binding B cells detected after immunization: 1) the TTCF binds TTCF-specific B cells that have undergone major clonal expansion; 2) TTCF binds via a non-clonal co-receptor expressed on B cells (e.g., antibody bound to FcγRIIb); and 3) B cells in immunized mice express a TTCF receptor. We are currently addressing these hypotheses experimentally.
The timing of stimulation and IL-2 signaling regulate secondary CD8 T cell responses (LYM5P.701)
Authors: Khan Shaniya , Martin Matthew , Starbeck-Miller Gabriel , Xue Hai-Hui Howard , Harty John , Badovinac Vladimir
Published: January 2023

Memory CD8 T cells provide protection to immune hosts by eliminating pathogen-infected cells during re-infection. While parameters influencing the generation of primary (1o) CD8 T cells are well established, the factors controlling the development of secondary (2o) CD8 T cell responses remain largely unknown. Here, we explored the mechanisms involved in the generation and development of 2o memory (M) CD8 T cells. We observed that the time at which 1o M CD8 T cells recognize Ag and become activated impacts their fate and differentiation into 2o M CD8 T cells. Late-entry of 1o M CD8 T cells into an immune response not only facilitated the expression of transcription factors associated with memory formation in 2o effector CD8 T cells, but also influenced the ability of 2o M CD8 T cells to localize within the LN, produce IL-2, and undergo Ag-driven proliferation. The timing of stimulation of 1o M CD8 T cells also impacted the duration of expression of the high-affinity IL-2 receptor (CD25) on 2o effector CD8 T cells and their sensitivity to IL-2 signaling. Importantly, by blocking or enhancing IL-2 signaling in developing 2o CD8 T cells, we confirmed the role of IL-2 in controlling the differentiation of 2o CD8 T cell responses. Thus, our data suggest that the process of 1o M to 2o M CD8 T cell differentiation is not fixed and can be manipulated, a notion with relevance for the design of future prime-boost vaccination approaches.