bluuebunny/crossref_metadata_2025_split huggingface dataset:
DNA origami: single cell analysis of T cell receptors without single cell sorting. (LYM7P.719)
Authors: Schoettle Louis , Wei Xixi , Garcia-Neurer Marlene , Zarnitsyna Veronkia , Antia Rustom , Yan Hao , Blattman Joseph 
Published: January 2023

Abstract
The immune system must be able to recognize virtually any pathogen (diversity) while maintaining enough cells specific for each pathogen in order to mount an effective response (protection). T cells generate diversity by imprecise joining of gene segments to generate alpha/beta heterodimeric receptors. Linking sequence information for TCRα and TCRβ from individual cells has been problematic due to the cost of single cell sorting and inadequate molecular approaches for linking mRNA encoding these proteins. We have developed novel DNA origami nanostructures to capture and protect both TCRα and TCRβ mRNA from individual cells, which can then be physically linked via a unique dual-primed reverse-transcription and ligation reaction to generate single amplicons containing both TCR from individual cells for use in next generation sequencing. We have demonstrated high efficiency transfection and recovery of DNA origami, optimized methods for purification with bound TCR mRNA, and validated this approach with transgenic T cells expressing a known TCR sequence. Surprisingly, we find that &lt;1% of TCR are shared even between genetically identical individuals, challenging the idea of “public” TCR. This approach is directly amenable to single cell analysis of other immune receptors (or other species) by relatively simple modifications of the origami sequences, and could be applied to virtually any heterogeneous cell population for which sequence information on any two genes is required.
“Natural” Th17 cells control Candida albicans infections in the oral mucosa (MUC7P.749)
Authors: Conti Heather , Peterson Alanna , Hernandez-Santos Nydiaris , Brane Lucas , Huppler Anna , Whibley Natasha , Hamilton Kristia , Garg Abhishek , Bishu Shrinivas , Simpson-Abelson Michelle , Mamo Anna , Artis David , Thomson Angus , Watkins Simon , McGeachy Mandy , Gaffen Sarah 
Published: January 2023

Abstract
Oropharyngeal candidiasis (OPC) is an opportunistic mucosal infection caused by the commensal fungus Candida albicans. To date, there is a paucity of research on oral mucosal immunity in general and to fungi in particular. Humans with rare mutations have revealed a vital protective role of IL-17 signaling to OPC. It has long been assumed that conventional “iTh17” cells produce IL-17. However, many innate populations also produce IL-17, but their role in oral immunity is unknown. Since rodents are naïve to C. albicans, we evaluated OPC in a murine model. T cell receptor rearrangement is required for immunity, since Rag1-/-, SCID and IL-7Rα-/- mice are susceptible to OPC. However, iTh17, NK and NKT cells were dispensable. We identified a CD3+CD4+CD44hiTCRβ+ tissue-resident population in tongue that produces IL-17 rapidly upon Candida exposure. These were phenotypically identical to ‘natural Th17’ cells, a recently described, thymically derived innate lymphocyte population whose function is highly enigmatic. Development of nTh17 and iTh17 cells involves differential AKT/mTOR pathways, with nTh17 cells requiring mTORC2, and iTH17 cells requiring mTORC1. Consistently, mice deficient in the mTORC2 subunit Rictor were highly susceptible to OPC, whereas mice treated with rapamycin, which selectively targets mTORC1, were similar to WT mice. Therefore, innate immunity to Candida requires nTh17 cells, whose presence in the oral mucosa ideally positions them to control oral pathogens.
Identifying lymphoma antigen receptor sequences by immune repertoire profiling (TUM2P.915)
Authors: Sherwood Anna , Robins Harlan , Fromm Jonathan , Greisman Harvey , Sabath Daniel , Emerson Ryan , Rieder Mark , Wood Brent , Wu David 
Published: January 2023

Abstract
Neoplastic T and B cells rapidly expand, leading to T and B-cell receptor (TCR and BCR) repertoires dominated by one rearrangement. However clones reacting to an antigen also expand. Differentiating high-frequency reactive clones from lymphomas is important for both diagnosing and monitoring lymphomas. To document the diverse repertoire of TRB and IGH CDR3 chains, we developed a method to deeply sequence the CDR3 rearrangements using high-throughput sequencing (HTS). This technology provides a potential opportunity to identify and monitor neoplastic clones during the course of therapy. To demonstrate the potential of HTS of T and B-cell receptors to contribute to diagnosing and monitoring neoplasia in mature lymphomas; we amplify the TCR repertoire of 98 and the BCR repertoire of 60 index samples to identify high-frequency TRB or IGH rearrangements. Concurrently, we sequence the TRB and IGH repertoire in control blood, bone marrow, and reactive lymph tissue. Clones are classified as neoplastic if occurring at a proportion greater than 7 standard deviations above the mean frequency of the most abundant rearranged TRB or IGH in control samples. Using these criteria, Eighty-four percent of index samples have a tractable rearrangement. We find that for most disease diagnoses, high-throughput sequencing identifies a tractable clone and T-cell and B-cell receptor repertoire analysis may be useful for clinical laboratory evaluation of patients with T and B-cell neoplasms.
IL-15 enhances the anti-tumor effect of human tumor antigen-specific T cells by cellular senescence delay (TUM2P.880)
Authors: Weng Jinsheng , Chu Fuliang , Baio Flavio , Kim Sung-Doo , Li Haiyan , He Jin , Qian Jianfei , Yang Jing , Watowich Stephanie , Yi Qing , Kwak Larry 
Published: January 2023

Abstract
Optimal expansion protocol for adoptive T-cell therapy often include interleukin (IL)-15, however, the mechanism by which IL-15 improves the in vivo anti-tumor effect of T cells remains to be elucidated. Using T cells generated from HLA-A2+ normal donors against novel T-cell epitopes derived from the U266 myeloma Ig light chain V-region (idiotype) as a model, we confirmed that T cells cultured with IL-15 provide more potent anti-tumor effects than IL-2-cultured T cells, after adoptive transfer in vivo into immunodeficient hosts. This effect was associated with delayed/reversed senescence in IL-15 expanded idiotype-specific memory CD8 T cells. In vitro experiments revealed IL-15 mediated senescence delay/reversal through JAK3-STAT5 and IL-2 receptor βγ signaling pathway. Specifically, we found DNA damage molecules MDC1, 53BP1, and ATM are the direct targets of IL-15 signaling and STAT5 represses the promoter activity of these genes through the competition with STAT3 to bind to the STATs sites. Thus, our study provides the first evidence for IL-15-inducing the repression of transcription of DNA damage molecules and senescence delay in human memory CD8 T cells.
MicroRNA-25 promotes gastric cancer migration, invasion and proliferation by directly targeting transducer of ERBB2, 1, and predicts poor patient survival (TUM2P.882)
Authors: Guo Gang 
Published: January 2023

Abstract
Gastric cancer (GC) is the most common tumors and the molecular mechanism underlying tumor metastasis is still largely unclear. Here, we show that miR-25 was overexpressed in plasma and primary tumor tissues of GC patients with TNM stage (III or IV) or lymph node metastasis. MiR-25 inhibition significantly decreased the metastasis, invasion and proliferation potential of GC cells in vitro, and reduced their capacity to develop distal pulmonary metastases and peritoneal dissemination in vivo. Furthermore, miR-25 repressed transducer of ERBB2, 1 (TOB1) expression by directly binding to the 3’-UTR, and the inverse correlation was observed between the expressions of miR-25 and TOB1 mRNA in primary GC tissues. Moreover, the loss of TOB1 increased the metastasis, invasion and proliferation of GC cells, and the restoration of TOB1 led to the metastasis, invasion and proliferation suppression. The receiver operating characteristics (ROC) analysis yielded an area under the curve (AUC) value of 0.7325 in distinguishing the GC patients with death from those with living. The analysis of optimal cutoff value revealed significant difference of patients’ survival between GC patients with high plasma concentrations of miR-25 (&gt; 0.2333 amol/μL) and those with low (&lt; 0.2333 amol/μL). Taken together, miR-25 promotes GC progress by directly down-regulating TOB1 expression, and the high concentrations of miR-25 in plasma of GC patients predict poor patient survival.
Investigation of the spatial heterogeneity of specific immune cell phenotypes in the tumor microenvironment of follicular lymphoma (TUM2P.884)
Authors: Mansfield James , Nelson Lili , Rose Chris , van der Loos Chris , Byers Richard 
Published: January 2023

Abstract
Tumor-infiltrating lymphocytes (TILs) are present in the tumor microenvironment of many cancers. Increased levels of regulatory T cells (Tregs) are associated with poorer prognosis in some cancers. An understanding of both the phenotype and spatial distribution of TILs within the tumor microenvironment is important in understanding their impact on the tumor. We present a method for analysing the distributions of CD3+FOXP3+ Treg and CD3+CD69+ activated T cells (Tact) in follicular lymphoma using a multispectral imaging (MSI) and automated analysis approach. A spatial distribution analysis was used to determine whether the patterns of T cells, Tregs and Tacts were prognostically significant. A single section of a TMA containing follicular lymphoma cores in triplicate from 40 subjects was stained for CD3, FOXP3, CD69 and hematoxylin. Each core was imaged using MSI. CD3+ TILs were located using automated image analysis. The FOXP3 and CD69 status of each CD3+ TIL was determined and the spatial distributions of the Tregs and Tacts was analyzed. Kaplan-Meier analysis demonstrated favorable outcome with those patients with higher numbers of each of CD3+, Tregs and Tacts. A diffuse spatial pattern of both Tregs and Tacts was associated with a favorable outcome. This is a general method capable of imaging, defining and measuring the effect on patient outcome of the spatial patterns of multiple cellular phenotypes in the tumor microenvironment.
Pregnancy increases the IgG4 and decreases the IgG1 response to influenza vaccine (VAC2P.935)
Authors: Schlaudecker Elizabeth , Way Sing , Finkelman Fred 
Published: January 2023

Abstract
Although the increased morbidity and mortality of influenza during pregnancy provide a compelling reason to vaccinate pregnant women, pregnancy is associated with a decreased hemagglutination inhibition (HAI) response to influenza vaccine. We hypothesized that in addition to this quantitative reduction, pregnancy-associated increases in IL-4, IL-10 and IL-13 secretion might promote production of IgG4 over the better complement- and Fc receptor-activating isotypes, IgG1 and IgG3; such a change might decrease antibody-dependent killing of virus. To begin to evaluate this hypothesis, we compared IgG1, IgG3 and IgG4 anti-H1N1 titers by ELISA in sera obtained from 50 pregnant and non-pregnant 18-39 year old women 24-31 days after vaccination. The IgG1:IgG4 ratio was significantly decreased in pregnant vs. non-pregnant women (29.0±35.0 vs. 69.3±94.1 (mean ± SD), p=0.04). Both HAI (p=0.017) and IgG1 (p=0.047) titers were decreased in trimesters 2 and 3 vs. trimester 1. These changes could be quite marked: 28% of women immunized during trimesters 2 and 3 had a combination of high IgG4/low IgG1 titers that was not seen in any non-pregnant or first trimester vaccinated women. Our results suggest that cytokine-mediated qualitative and quantitative changes in the anti-influenza antibody response may decrease protection in a considerable percentage of women vaccinated during the last 2/3 of pregnancy, which, in turn, suggests a need to consider changes in vaccination for this population.
Broad spectrum immunity against influenza viruses elicited by intranasal vaccination with a new recombinant subunit vaccine (VAC2P.924)
Authors: Li Junwei , Arévalo Maria , Chen Yanping , Smith Jacob , Pasadas Olivia , Zeng Mingtao 
Published: January 2023

Abstract
Frequent mutation of influenza viruses keep vaccinated and non-vaccinated populations vulnerable to new infections, causing serious burdens to public health and the economy. Vaccination with universal influenza vaccines would be the best way to effectively protect people from infection caused by mismatched or unforeseen influenza viruses. Presently, there is no FDA approved universal influenza vaccine. In this study, we expressed and purified a fusion recombinant protein CTB-3×M2e-HA2 which comprises of influenza matrix 2 protein ectodomain peptides, a centralized influenza hemagglutinin stem region, and cholera toxin subunit B. Intranasal vaccination of BALB/c mice with this novel artificial antigen resulted in potent humoral immune responses, including induction of antigen specific IgA and IgG, and broad protection against infection by multiple influenza viruses including mouse adapted H1N1 PR8, 2009 pandemic H1N1, and H3N2 influenza A viruses. Furthermore, our results demonstrated that when used as a mucosal antigen, cholera toxin subunit B improved antigen-stimulated T cell and memory B cell responses. Therefore, the candidate new nasal influenza vaccine has potential to be used in the event of a pandemic flu crisis, and provide relief to the public health burden.
CD40-targeted nucleoprotein provides enhanced immune response and protection against influenza virus (VAC2P.927)
Authors: Hashem Anwar , Gravel Caroline , Tocchi Monika , Jaentschke Bozena , Fan Xingliang , Chen Ze , Li Changgui , Junzhi Wang , Li Xuguang 
Published: January 2023

Abstract
Influenza nucleoprotein is a highly conserved antigen and a candidate universal vaccine against flu. However, NP-based vaccines can only afford weak protective immunity compared to HA. Using CD40 ligand (CD40L), a key regulator of the immune system, as both a targeting ligand and a molecular adjuvant, we show that single immunization with recombinant adenovirus carrying a fused gene encoding for secreted NP-CD40L fusion protein provided robust and long-lasting protection against influenza in normal mice. It enhanced B cell responses by inducing early and persistent B cell germinal center formation, accelerated immunoglobulin isotype-switching and TH1-skewed NP-specific immune response. Moreover, it significantly boosted primary and secondary NP-specific cytotoxic T-lymphocyte (CTL) activity and multiple cytokine producing-CD8+ T cells. Transfer of sera or CD8+ T cells from immunized to naïve mice showed that targeting NP to CD40-expressing cells augmented the role of both NP-specific antibodies and CTLs in protection compared to untargeted NP. Importantly, it afforded effective protection in CD40L and CD4 deficient mice, confirming that the induced protection is CD40L-mediated and CD4+ T cell-independent.
Novel IgM-derived antibody-drug conjugate selectively kills chronic lymphocytic leukemia cells through binding of Fcμ-receptor leading to rapid internalization and intracellular cytotoxic payload delivery (VAC3P.953)
Authors: Skarzynski Martin , Vire Bérengère , Thomas Joshua , Nelson Christopher , David Alexandre , Aue Georg , Burke Terrence , Rader Christoph , Wiestner Adrian 
Published: January 2023

Abstract
The IgM Fc receptor (FcμR) is a previously unexploited target for monoclonal antibody therapy and antibody drug conjugate (ADC) therapy of B-cell chronic lymphocytic leukemia (CLL). While anti-FcμR IgG binding does not cause FcμR internalization, IgM bound to FcμR are rapidly internalized (&gt;80% internalized within 5 minutes). FcμR is overexpressed in CLL with only limited expression on normal B, T, and NK cells. In our study, 29 out of 30 CLL patient samples (97%) evaluated by flow cytometry had high FcμR expression by CLL cells (mean(SD)=89.1(7.1)%) and the remaining patient had moderate CLL FcμR expression (57.9%). In contrast, FcμR expression by T cells (mean(SD)=8.9(5.2)%) was significantly lower (p&lt;0.0001). We developed a FcμR targeting ADC consisting of the CH2-CH3-CH4 IgM regions conjugated to monomethyl auristatin F. Flow cytometric analyses of CLL patient samples showed potent ADC mediated killing of CLL cells (mean(SD)=78.8(10.1)%, p&lt; 0.0001 versus scaffold treated control) but not T cells (mean(SD)=8.4(3.8)%, p=0.3210 versus scaffold treated control). The ADC also greatly reduced tumor burden in NOD/SCID/IL-2Rγnull mice xenografted with 4 CLL patient samples (mean reduction(SD)=74(20)% in blood and 65(19)% in spleen, n=30). In summary, FcμR is a promising new target for therapy of CLL and possibly other malignancies. Our preclinical results provide translational evidence for the clinical utility of IgM-derived protein scaffolds to target FcμR expressing cells.
Macrophages stimulated with mollusk hemocyanins present a different expression pattern of proinflammatory cytokines and chemokines (VAC3P.956)
Authors: Becker María , Zhong Ta-Ying , Arancibia Sergio , Born Raimundo , Tampe Ricardo , Del Campo Miguel , Manubens Augusto 
Published: January 2023

Abstract
Hemocyanins inoculated in mammals induce a Th1-dominant response with beneficial clinical outcomes, being used as carrier/adjuvant in vaccines and as immunostimulant for the treatment of bladder cancer. However, its effects during the early phases of the immune response are unknown. We hypothesized that when hemocyanins are incorporated by antigen presenting cells, they induce an inflammatory milieu, producing a bystander effect, key event in its non-specific immunostimulatory effects. Here, in vitro cultures of murine peritoneal macrophages were incubated with different hemocyanins from M. crenulata (KLH) or C. concholepas (CCH) or F. latimarginata (FLH), to study the correlation between mRNA and protein levels from a panel of proinflammatory cytokines and chemokines, using qPCR and ELISA. The results showed that hemocyanins were endocyted and slowly processed by macrophages, stimulating the expression of IL-6, IL-12p40 and TNF-α at different times and intensities. FLH and KLH but not CCH at 24 h, induced an increased in the expression of chemokines related to leukocyte extravasations, such as CCL-3, CXCL-1 and CXCL-5. Moreover, hemocyanins kept down-regulated key Th2 cytokines such as IL-4 and TGF-β2. We concluded that each hemocyanin can stimulate different kinetic patterns of proinflammatory cytokines and chemokines expression in macrophages, suggesting different signaling pathways, which can be explained in part, by its structural differences.
Natural and anti-PD-L1 induced tumor immunity in a novel ovarian cancer mouse model for human mucin 1 (MUC1) (VAC3P.943)
Authors: Zhang Lixin , Tirodkar Tejas , Elishaev Esther , Mony Jyothi , Brozick Jone , Edwards Robert , Vlad Anda 
Published: January 2023

Abstract
MUC1 is a large cell surface glycoprotein overexpressed by almost all types of adenocarcinomas, including those originating in the ovaries. We recently generated a triple transgenic MKP mouse model that carry conditional Kras mutations, Pten deletion and express human MUC1 as self. Upon AdCre injection under the ovarian bursa, the MKP mice progress to endometrioid ovarian tumors. Using a freshly isolated, orthotopic ovarian tumor from a MKP mouse, we generated an ovarian cancer cell line MKP-T-2F8 (or simply 2F8) that triggers multiple MUC1 expressing intraperitoneal (IP) tumors. At baseline, the cells express low PD-L1 but expression increases after in vivo growth. Using the 2F8 transplantable tumor model, we tested in vivo the therapeutic effectiveness of anti-PD-L1 antibody (10F.9G2). MUC1.Tg or wild type mice were IP injected with 8x10E5 of 2F8 cells, followed by three doses of 200ug of anti-PD-L1 (or isotype control) antibody, administered IP every 2 weeks, starting 3 weeks post-tumor challenge. The anti-PD-L1 treated MUC1.Tg mice showed significantly increased survival (p= 0.02), with no evidence for peritoneal tumor. In contrast, wild type mice which grow 2F8 tumors and develop spontaneous anti-MUC1 antibodies in high titers, did not respond to anti-PD-L1 therapy. These results demonstrate that targeting PD-L1 is a viable therapeutic strategy in ovarian cancer; however, tumor-bearing hosts with Th2-driven immune imbalance may not benefit from PD-L1 blockade.
Scavenger receptor CD204+ macrophages play an immunoregulatory role in hepatic viral infection (VIR1P.971)
Authors: Labonte Adam , Sung Sun-Sang , Hahn Young 
Published: January 2023

Abstract
Macrophages (MΦ) are important for the activation and direction of innate and adaptive immune responses, however their role in liver infection is particularly poorly understood, in part due to the presence of functionally distinct subsets within the population. The MΦ scavenger receptor CD204/SRA is a marker for alternatively activated (M2) MΦ, which have anti-inflammatory properties and participate in wound healing and tissue remodeling. To understand the function of CD204+ MΦ on regulating liver inflammation, we examined the activation and differentiation of MΦ in a murine model of hepatic viral infection. Our studies revealed that CD204 was upregulated on circulating and liver-resident MΦ following tail vein injection of adenovirus or MCMV. CD204+ macrophages appear to express high levels of PD-L1 and IL-10, suggesting an alteration of MΦ phenotype. Interestingly, virally infected CD204-deficient mice had less liver CD8+ T cells than their wild type counterparts and these T cells produced less IFNγ and granzyme B. In contrast, liver NK and NKT cell populations in knockout and WT mice were similar in size, but NK cells from CD204 deficient mice displayed increased granzyme B staining following infection. These data indicate that CD204+ MΦ play a critical role in modulating the immune response to hepatic viral infection.
A scalable RNA RT-PCR based assay to quantify latency disruption in HIV-1 infected cells from patients on antiretroviral therapy (VIR1P.1001)
Authors: Hataye Jason , Casazza Joseph , Ambrozak David , Boritz Eli , Yamamoto Takuya , Douek Daniel , Koup Richard 
Published: January 2023

Abstract
Antiretroviral therapy suppresses HIV-1 replication but virus persists in stable reservoirs. A facile way to quantify cells in such reservoirs is HIV DNA PCR, however most of these cells are unlikely to ever produce replication-competent virus. On the other hand, viral outgrowth assays provide virologic realism, but are resource intensive. Detection of RNA by RT-PCR provides some virologic realism while allowing for high throughput. We present a method in which flow cytometry purified CD4+ T cells from patients on antiretroviral therapy are placed in limiting dilution culture for 4 days. The cells are stimulated with antibodies to CD2, CD3, and CD28 and viral replication is inhibited with efavirenz. Viral RNA is purified from the culture media using a paramagnetic nanoparticle based method that is scalable for robotics. Real time quantitative RT-PCR is performed with an HIV gag primer and probe set. This assay detects as few as 47 copies of HIV RNA induced from a single cell from the ACH-2 cell line. Using resting memory CD4+ T cells from patients on antiretroviral therapy, the frequency of induced HIV RNA production varied from 1 in 40,000 cells to fewer than 1 in 1,000,000. There was no correlation between frequency of HIV RNA producing cells and frequency of proviral DNA. This assay represents a scalable method for quantifying the frequency of HIV producing cells from patients with viral suppression and for evaluating reservoir elimination strategies for such patients.