bluuebunny/crossref_metadata_2025 huggingface dataset:
ROR1 directed delivery of FTY720 derivative OSU-2S mediates potent cytotoxic activity against chronic lymphocytic leukemia but not normal B cells in-vitro and in ROR1 expressing transgenic CLL mouse model in-vivo (P3275)
Authors: Muthusamy Natarajan , Mao Yicheng , Frissora Frank , Mani Rajeswaran , Wang Jiang , Wu Yun , Yu Bo , Baskar Sivasubramanian , Rader Christoph , Phelphs Mitch , Chen Ching-Shih , Lee Robert , Lee L. , Byrd John
Published: January 2023

ROR1, a member of the receptor tyrosine kinase-like orphan receptor family is expressed in chronic lymphocytic leukemia (CLL) but not normal B cells. We describe here a novel anti-human ROR1 antibody (2A2-IgG) based immunoliposomal delivery system for CLL therapy with improved specificity in vitro and in vivo. 2A2-IgG immobilized onto the surface of the liposomes (2A2-IgG-ILP), was evaluated for targeted delivery of a recently described novel phosphatase activator, OSU-2S. 2A2-IgG-ILP-OSU-2S demonstrated improved cytotoxicity in ROR1 positive CLL cells but not in normal B cells in-vitro. To validate the in-vivo efficacy of the ROR1 directed 2A2-IgG-ILP-OSU-2S, we developed transgenic mice expressing hROR1 on B cells (hROR1 Tg). Crossing the hROR1 Tg mice to the previously described Eμ-Tcl1 CLL mice resulted in double Tg mice with progressive CLL like disease characterized by accumulation of CD5+CD19+ROR1+ B cells. 2A2-IgG-ILP-OSU-2S exhibited selective binding and delivery of OSU-2S to ROR1+ but not ROR1- B cells in-vitro and elimination of circulating ROR1+ B cells in-vivo. Additionally, preliminary pharmacokinetic analysis of the targeted and non-targeted ILP-OSU-2S in the ROR1 transgenic mouse revealed altered pharmacokinetic profiles with 2A2-IgG targeted formulation. These studies validate the ROR1 targeted immunoliposomal formulations and the novel transgenic mouse models for evaluation of therapeutic agents directed to ROR1 positive B cell malignancies.
IL-1 and IFN-gamma differentially program human dendritic cells exposed to beta-glucan via IkappaB-zeta regulation (P6279)
Authors: Cardone Marco , Dzutsev Amiran , Li Hongchuan , Gerosa Franca , Winkler-Pickett Robin , Provezza Lisa , Riboldi Elena , Orr Selinda , Steinhagen Folkert , Wewers Mark , McVicar Daniel , Anderson Stephen , Goldszmid Romina , Lyakh Lyudmila , Trinchieri Giorgio
Published: January 2023

The molecular networks controlling the DC programming in response to C-type lectin receptor agonists including beta-glucan, a fungal and bacterial component and ligand for the Dectin-1 receptor, are still poorly characterized. We studied these immunological aspects by comparing the response of human mono-DCs to the Dectin-1 ligand, beta-glucan, and to the TLR4 ligand, LPS using a gene expression/perturbation approach. Gene expression analysis of mono-DCs stimulated by beta-glucan identified IL-1, TNF, and type I IFN encoded by “early/immediate” type genes as predicted regulators of the beta-glucan-induced “late” transcriptional response. A perturbation analysis revealed that endogenous IL-1, via the nuclear factor IkappaB-zeta, selectively promoted the expression of beta-glucan-induced “late” genes such as those encoding IL-6 and IL-23, thus programming DCs to initiate Th17 responses. TNF and type I IFN were found to modulate the response to both beta-glucan and LPS. IFN-gamma counteracted the activity of IL-1 and reprogrammed human mono-DCs to promote Th1-like cells rather than Th17 in response to beta-glucan. Our data provide evidence that IL-1 and IFN-gamma promote distinct adaptive immune responses against the fungal and bacterial component beta-glucan by differentially modulating the DC programming. They also suggest potential strategies to modulate the immunity against beta-glucan containing pathogens.
Cytotoxic effector CD4 T cells express NKG2C/E and are defined by a unique program dependent on Blimp-1. (IRC8P.482)
Authors: Vong Allen , Marshall Nikki , Kuang Yi , Berg Leslie , Swain Susan
Published: January 2023

CD4 T cells with cytotoxic activity can be generated during many immune responses to acute viral infections and they can contribute to CD4-mediated viral control. During the peak of the CD4 T cell response to an acute infection of influenza A/PR8 [IAV], cytotoxic CD4 T cells [ThCTL] are found in the lung. The cytotoxicity they mediate is MHC II-restricted and perforin-dependent. We find the lung ThCTL express NKG2C and E that are members of the killer cell lectin-like receptor family [KLR]. Although the ThCTL cytotoxicity is strictly antigen dependent, we show that NKG2C/E can costimulate the cytotoxic activity of ThCTL in vivo, since target cells lacking the ability to present peptide on Qa-1b, which binds to NKG2C/E, are significantly protected from lysis by ThCTL during IAV infection. The ThCTL express a unique pattern of molecules including high levels of granzyme B, PSGL-1, PD-1 and CD27 as well as transcription factors Eomes and Blimp-1. We find that Blimp-1 is critical for the generation and differentiation of this subset, since a CD4 T cell intrinsic defect in Blimp-1 leads to a broad reduction of most of the ThCTL program including cytotoxic activity and migration to the lungs as well as expression of NKG2C/E, granzyme B, PSGL-1 and CD27. These features help to define a distinct ThCTL cell subset generated under distinct conditions during an acute viral infection that has novel target specificity and thus likely occupies a unique functional niche.
Streptococcus pneumoniae specific Th17 memory immunity provides cross protection against invasive pneumococcal diseases in mice. (IRC9P.701)
Authors: Wang Yan , Tian Ying , Jiang Bin , Weiser Jeffery , Shen Hao
Published: January 2023

Pneumonia caused by Streptococcus pneumoniae (Sp) remains a leading cause of serious illness and numerous deaths in children and elderly worldwide. Current pneumococcal vaccine is effective in preventing colonization by inducing serotype-specific antibodies. However, there is an increasing prevalence of infection by serotype strains not included in the vaccine; this highlights the need for a universal vaccine that protects against all serotypes. In current study, we found that mice intranasal immunized with Sp were protected against challenge with a different serotype Sp strain and cleared bacteria from the lung by day 2 post-challenge, and exhibited less apoptosis and tissue damage in lung. Sp infection in lung resulted in a tremendous CD4+T cell expansion and activation that consisted of mostly IL-17 producing Th17 cells but also IFNγ producing Th1 cells. Adoptive transfer of T cells from Sp immunized mice provided cross protection with a dominant Th17 recall response in the lung from donor CD4+ memory T cells. Furthermore, adoptive transfer of Sp specific CD4+ memory T cells provided cross protection against pneumonia and bacteremia, which can be blocked by IL-17 neutralizing antibody and transfer Sp memory CD4+ T cells from IL-17 knockout mice. Our results suggest that Sp memory Th17 cells played a key role in providing broad protective immunity against invasive Sp infection in a serotype independent manner.
POU2AF1 arranges systemic distribution of follicular helper T cells (IRC10P.466)
Authors: Yamashita Keiji , Mitsuhashi Yukari , Nagaya Tomonori , Jistukawa Sumito , Kamekura Ryuta , Seki Nobuhiko , Kumagai Ayako , Kawata Koji , Himi Tetsuo , Ichimiya Singo
Published: January 2023

Follicular helper T (Tfh) cells are a distinctive subset of CD4+ T helper cells, which play a critical role to establish specific humoral immunity by helping the germinal center formation of B cells. Currently, it is known that Tfh cells are maintained by BCL-6, MAF, and BATF; however, a mechanism managing Tfh cells is not fully clarified yet. To further dissect a regulator of Tfh cells, we employed transcriptome analysis of human tonsillar Tfh cells and thymic naïve T cells as a control. Results demonstrated that, in addition to BCL-6, MAF, and BATF, Tfh cells highly expressed POU2AF1 (BOB.1 or OBF1) at transcript and protein levels. POU2AF1 is a transcriptional coactivator associated with either OCT1 or OCT2 through recognition of POU domains and assists B cells to produce specific antibodies. Analysis using Pou2af1-deficient mice showed that the expression levels of Pou2af1 did not seem to correlate the levels of Cxcr5,Icos,Tigit and Bcl-6 in Tfh cells. Mix cell culture experiments further revealed no functional differences in between Pou2af1-negative Tfh cells and control Tfh cells. But it was of our interest that, after stimulation with foreign antigens, the frequencies of Tfh cells in immune tissues and peripheral blood of Pou2af1-deficient mice were altered in comparison to those of wild-type mice. Taken together, these suggest that POU2AF1/Pou2af1 might have a role to regulate spreading manner of Tfh cells over the body from local sites of immune response.
Myeloid dendritic cells maintain Th17 lymphocyte responses during the obstructive phase of experimental biliary atresia (IRC10P.471)
Authors: Lages Celine , Simmons Julia , Maddox Avery , DiPasquale Betsy , Shanmukhappa Shiva Kumar , Chougnet Claire , Miethke Alexander
Published: January 2023

Biliary atresia (BA) is a fibro-inflammatory obstruction of the extrahepatic biliary tree in newborns. It leads to rapid biliary fibrosis and is the most common indication for pediatric liver transplantation. In a model of rhesus rotavirus (RRV)-induced murine BA, plasmacytoid dendritic cells (DC) initiate NK-cell mediated hepatobiliary injury at 3 days post infection (dpi). We hypothesize that myeloid (m)DC coordinate T-cell driven injury during the later obstructive phase of BA. Following RRV injection into CD11c-DTR GFP pups on day 1 of life, frequency of hepatic DC increased 6fold at 12dpi compared with non-infected controls. Expansion of mDCs was accompanied by accumulation of activated CD4 cells in the liver. Plasma IL17A/F levels were increased at 8 and 12dpi (p<0.01) and hepatic IL17A mRNA increased 9fold between 8 and 12dpi. In vitro coculture of DC from 12dpi pups with naïve CD4 cells induced IL17A/6 production. DC-depletion following RRV infection with diphtheria toxin reduced the number of hepatic CD4 cells by 3fold which was associated with 2fold decrease in hepatic IL17A mRNA expression and plasma Th17-associated cytokine levels (IL-17F/6) at 12dpi. Importantly, diminished Th17 responses correlated with reduced plasma total bilirubin levels (6.1 vs 11.3mg/dL in control; p<0.01), a biomarker for biliary obstruction. We conclude that mDC maintain hepatic Th17 responses and regulate cholestatic liver injury during the obstructive phase of experimental BA.
Gene targeting of RhoA reveals its essential role in thymocyte development and metabolism (LYM7P.718)
Authors: Guo Fukun , Zhang Shuangmin , Konstantinidis Diamantis , Yang Jun-Qi , Kalfa Theodosia , Zheng Yi
Published: January 2023

RhoA GTPase is an intracellular signal transducer capable of regulating a wide range of cell functions including cytoskeleton dynamics, proliferation, and survival. In T lymphocytes, studies by using dominant negative mutant or C3 transferase expressing transgenic mice suggest that RhoA is involved in TCR signaling and related T cell functions such as polarization, migration, survival, and proliferation. To date, the physiological role of RhoA in T cell development remains unclear. In this study, we have achieved T cell-specific deletion of RhoA and found that RhoA gene disruption caused (1) a drastic decrease in the numbers of all thymocyte subsets including CD4-CD8- double negative (DN), CD4+CD8+ double positive (DP), CD4+CD8- single positive (SP), and CD4-CD8+ SP T cells, (2) an impaired β-selection at DN stage and positive selection at DP stage associated with altered pre-TCR and TCR signaling transduction, respectively, (3) a diminished thymocyte proliferation which could be due to impaired cytokinesis, (4) an increased apoptosis, and (5) an enhanced mitochondrial function as manifested by increased oxidative phosphorylation, glycolysis, mitochondrial membrane potential, mitochondrial numbers, mitochondrial DNA and ROS production. Thus, RhoA critically regulates T cell development by impacting on β-selection, positive selection, cell proliferation and survival through mediating pre-TCR signaling, TCR signaling, cytokinesis, and metabolism.
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

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 <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

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

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

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

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 (> 0.2333 amol/μL) and those with low (< 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

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

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

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

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

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 (>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<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< 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

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

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

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

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.
Characterization of human soluble Tim-3 and its role in HIV infection (VIR1P.1002)
Authors: Clayton Kiera , Douglas-Vail Matthew , Medcalf Karyn , Chew Glen , Tandon Ravi , Ndhlovu Lishomwa , Ostrowski Mario
Published: January 2023

Chronic Human Immunodeficiency Virus Type-1 (HIV) infection results in a loss of HIV-specific CD8+ T cell effector function, termed “exhaustion”, which is mediated, in-part, by the co-inhibitory receptor T-cell immunoglobulin mucin domain-containing protein 3 (Tim-3). Like many other receptors, a soluble form of this protein has been described in human blood plasma. However, this soluble Tim-3 (sTim-3) is poorly characterized and it’s unclear whether it plays a role in HIV pathogenesis. Using a Tim-3 ELISA, we found that levels of sTim-3 were significantly elevated in HIV+ antiretroviral treatment naïve patient plasma compared to healthy controls or HIV+ antiretroviral treated patients, and significantly correlated positively with viral load but not with CD4+ T cell counts. To characterize this construct, we immunoprecipitated the protein from HIV+ patient plasma; western blotting analysis revealed that plasma sTim-3 is the approximate size of the Tim-3 ectodomain. Following detection of sTim-3 from activated PBMCs in culture, no alternatively spliced Tim-3 constructs were observed, however, levels of sTim-3 were abrogated with the inclusion of a matrix metalloproteinase, ADAM10, inhibitor. Further, we showed that this sTim-3 was produced from activated T cells treated with an ADAM10 agonist and was the approximate size of plasma sTim-3. Our results suggest that increasing levels of viral antigen result in increased Tim-3 shedding from the T cell surface during HIV infection.
Hepatitis B Virus X protein inhibits A20 expression and sensitizes TRAIL-induced apoptosis in hepatocyte cells (VIR1P.1006)
Authors: Shi Liyun , Zhang Hang , Kang Yanhua , Lu Zhe , Wang Huanhuan
Published: January 2023

Hepatitis B virus (HBV) infection is a crucial cause of hepatocyte death, cirrhosis, and hepatocellular carcinoma. However, the molecular mechanism causing the death of hepatocytes during HBV infection was not completed understood. A20, also named tumor necrosis factor alpha-induced protein 3 (TNFAIP3), is a key regulator in inflammation and immunity, and was identified as a target of miR-125a. Here, we report that miR-125a is induced by hepatitis B virus X protein (HBx) and leading to repression of A20. We also found HBx or miR-125a transfection of hepatoma cell line L-O2 significantly increased its sensitivity to TRAIL-induced apoptosis. The increasing of TRAIL sensitivity was associated with repression of A20 protein expression, and HBx activates caspase-8, due to the direct cleavage of RIP1 by A20-inhibition. We also found caspase-8 K63-linked polyubiquitination was repressed by HBx. Thus, our study indicates that HBx sensitizes hepatocytes to TRAIL-induced apoptosis through upregulating miR-125a and targeting A20 through caspase-8 activation and the regulation of RIP1 ubiquitination. So these results suggest that miR-125a and A20 represent key diagnostic markers and potential therapeutic targets for the prevention and treatment of HBV infection.
Natural killer cells suppress humoral immunity and development of neutralizing antibodies (VIR1P.962)
Authors: Rydyznski Carolyn , Daniels Keith , Karmele Erik , Brooks Taylor , Sutiwisesak Rujapak , Welsh Raymond , Waggoner Stephen
Published: January 2023

Pathogen-specific neutralizing antibodies (nAbs) are a key component of protective immunity after infection or vaccination. However, broadly-specific nAbs that can prevent infections with highly mutable viruses like HIV are rarely seen in infected patients and are poorly elicited by current vaccines. Therefore, new vaccine strategies are needed to enhance the induction of nAbs, potentially by augmenting the germinal center (GC) reactions that facilitate somatic hypermutation, affinity maturation and development of high-affinity nAbs. Here, we demonstrate that natural killer (NK) cells impair humoral immunity by contributing to a weak GC response and subdued generation of virus-specific nAbs after acute lymphocytic choriomeningitis virus (LCMV) infection of mice. The magnitude and duration of the LCMV-induced GC response was substantially reduced by NK cells, which constricted the number of both follicular helper T cells (Tfh) and GC B cells present in lymphoid tissues. NK cell-mediated suppression of GC responses was also observed in different stains of mice and after infection with a variety of dissimilar pathogens, suggesting that NK cell suppression of humoral immunity is a universal feature of infection. Notably, NK cell inhibition of the GC response during LCMV infection resulted in a markedly delayed and relatively weak nAb response. Thus, targeting of immunosuppressive NK cells could represent a revolutionary method to enhance the efficacy of future vaccine regimens.
Myeloid cell Arg1 and iNOS inhibit control of arthritogenic alphavirus infection by suppressing anti-viral T cells (VIR1P.965)
Authors: Burrack Kristina , Her Zhisheng , Gill Ron , Ng Lisa , Morrison Thomas
Published: January 2023

Mosquito-borne alphaviruses, including chikungunya virus (CHIKV) and Ross River virus (RRV), cause a debilitating, and often chronic, rheumatologic disease in humans. Utilizing mouse models that recapitulate many aspects of the human disease we found that arginase 1 (Arg1) and nitric oxide synthase 2 (iNOS) are highly induced in inflamed musculoskeletal tissues and infiltrating macrophages in RRV- and CHIKV-infected mice. Similar to myeloid-derived suppressor cells (MDSCs), these Arg1/iNOS-expressing macrophages suppress T cell proliferation ex vivo. Depletion of T cells restored RRV loads in musculoskeletal tissues of Arg1-deficient mice to similar levels as T cell-depleted WT mice, suggesting that Arg1+ suppressor cells inhibit T cell-mediated control of alphavirus infection. Moreover, myeloid cell Arg1 reduced T cell production of anti-viral cytokines and altered the activation phenotype of virus-specific T cells in inflamed muscle tissue, but not lymphoid tissue, of RRV-infected mice. Furthermore, the expression of Arg1 and several other MDSC-associated genes were significantly higher in PBMCs collected from CHIKV-infected patients compared to PBMCs from healthy controls. Expression of Arg1 remained elevated months after illness onset, and increased expression levels of Arg1 correlated with higher viremia. These studies suggest that CHIKV/RRV infection activates immunosuppressive monocytes/macrophages that antagonize virus control and may contribute to chronic disease.
Crossreactive Epstein-Barr virus (EBV)-influenza A virus (IAV) specific CD8 memory T cells during acute symptomatic IAV infection. (VIR2P.1022)
Authors: Mishra Rabinarayan , Gil Anna , Aslan Nuray , Luzuriaga Katherine , Selin Liisa
Published: January 2023

We previously showed that crossreactivity is common between IAV and EBV in HLA-A2+ patients during infectious mononucleosis. IAV-M1 specific CD8 T cells, along with expanded populations of IAV-M1/EBV-BRLF-1 and IAV-M1/EBV-BMLF-1 double-tetramer+ cells were detected directly ex-vivo in 5 HLA-A2+ patients. Altered IAV-M1, EBV-BRLF1 and -BMLF1 TCR repertoires were observed over the course of infection and in comparison to healthy donors. After culture, cells were sorted and analyzed by gene array in order to assess global changes in immune responses following different stimulations, either cognate or crossreactive, in different patient populations. M1- and BRLF1-specific cells had similar immune-response gene signatures, but the BMLF1-specific CD8 cells were more similar to the two-crossreactive populations. Crossreactive M1/BRLF1 cells from the BRLF1 line were different in their activation status than the BRLF1-specific cells, consistent with BRLF1 ligand stimulation of different gene activation profiles in these two populations. These results suggest that during symptomatic IAV infection there is an expansion of EBV/IAV crossreactive memory CD8 T cell responses. Ongoing studies are investigating whether EBV-IAV cross-reactive CD8+ T cells may contribute to immunopathology during acute IAV infection.
Increased PGD2 is associated with delayed dendritic cell maturation in neonates during respiratory viral infection (VIR2P.1031)
Authors: Shornick Laurie , Bhattacharya Somashubhra , Tumala Brunda
Published: January 2023

During respiratory viral infection in adults, inflammatory cytokines stimulate conventional dendritic cells (cDC) to increase expression of MHC class II molecules, co-stimulatory molecules, and chemokine receptors. Activated cDC then migrate from the lung to the draining lymph nodes where they present antigen to T cells. Our previous studies demonstrated that neonatal mice exhibit reduced inflammation and inflammatory cytokine expression during paramyxoviral infection. Thymic stromal lymphopoietin (TSLP), a cytokine known to participate in cDC activation, is expressed at significantly lower levels in neonatal mice during respiratory viral infection compared to adults. Thus, we hypothesized that cDC maturation and migration to lymph nodes may be delayed or impaired in neonates. Adult and two-day old neonatal C57BL/6 mice were inoculated intranasally with Sendai virus (500 pfu/g body weight). Adult cDC had increased expression of MHC Class II and CCR7 and had migrated to the lymph nodes on post-infection days 1 and 3. However, these events were delayed in neonates and did not reach adult levels until post-infection day 7. Interestingly, studies in geriatric mice suggest that high levels of Prostaglandin D2 (PGD2) may inhibit cDC maturation and migration to lymph nodes. We also observed higher PGD2 expression in neonatal bronchoalveolar lavage fluid compared to adults during paramyxoviral infection, and this may contribute to the impairment of DC maturation in neonates.
The role of miR-328 in respiratory diseases (INM3P.410)
Authors: Tay Hock , Kaiko Gerard , Mattes Joerg , Hansbro Philip , Foster Paul
Published: January 2023

Introduction: MicroRNAs (miRNAs) are small non-coding RNA that can bind to multiple target mRNA to repress protein production. Deregulation of miRNAs have been linked with pathogenesis of multiple human diseases. In immune cells, miRNAs regulate cell development, differentiation and production of inflammatory mediators. Aim: To investigate the roles of miRNAs in regulating immune cell function and its role in respiratory diseases. Methods: Lung miRNAs that were differentially expressed following non-typeable Haemophilus Influenzae (NTHi) challenge were identified by microarray. miRNA inhibitors (antagomirs) were used to investigate the role of miRNA in regulating bacterial clearance in in mouse models of immunosuppresion and emphysema. Results: Upon NTHi infection, 15 miRNAs were up-regulated while 49 were down-regulated by > 2.5 fold in lungs. In vitro, miR-328 knockdown reduced bacterial load and increased phagocytosis by macrophages and neutrophils. Transferred of macrophages or neutrophils (deficient in miR-328) to naïve recipient mice enhanced clearance of the bacteria in vivo. Similarly, inhibition of miR-328 by antagomirs promote NTHi clearance in mouse models of immunosuppresion and emphysema. Conclusion: : Our study identify a novel role for miR-328 in the regulation of phagocytosis and provide proof of principle that miRNA pathways can be targeted in the lung and offer a potential new anti-microbial approach for the treatment of respiratory infection.