bluuebunny/crossref_metadata_2025 huggingface dataset:
Transcriptome analysis of lung resident compared to spleen memory CD4 T cells reveals differential expression of genes controlling tissue egress (P5083)
Authors: Yudanin Naomi , Farber Donna
Published: January 2023

Memory T cells, generated after pathogen encounter, persist in diverse tissue sites, including lymphoid and mucosal tissues. We previously identified a population of memory CD4 T (CD4TM) cells that were retained in the lung, and mediated optimal protective responses to respiratory virus infection. Spleen-derived CD4 TM cells circulated in multiple sites and were not protective. Here, we investigated potential mechanisms for these differential migration patterns by whole transcriptome analysis of lung and spleen CD4 TM cells. We isolated RNA from lung and spleen CD4 TM generated in vivo for subsequent RNAseq analysis. Our analysis revealed a set of genes that are differentially expressed in lung compared to spleen CD4 TM cells. Using unbiased gene ontology clustering (GO), functional pathway analysis (KEGG), and gene interaction network mapping (STRING9.0) we identified a cohesive gene interaction pathway that functions in cell motility and tissue retention. Along with other genes involved in cell adhesion and migration, the selected candidates included Sphingosine-1-phosphate receptor (S1PR1), CD69, Kruppel-like factor (KLF)-2, and KLF6, all known to regulate lymphocyte egress from tissues. Upon re-activation, lung CD4TM upregulated CD69 and KLF6, and downregulated S1PR1 and KLF2 compared to spleen CD4TM. These findings suggest that the upregulation of these genes in lung but not in spleen memory CD4 T cells may contribute to their tissue-specific retention in the lung.
Concurrent analyses of host-pathogen transcriptomes in avian polymorphonuclear cells following exposure to Salmonella enterica serovar Typhimurium (P4233)
Authors: Swaggerty Christina , Chiang Hsin-I , Zhou Huaijun , Kogut Michael
Published: January 2023

Salmonella enterica serovar Typhimurium are facultative intracellular bacteria that cause disease in numerous species, and infections resulting from poultry products are a major source of foodborne disease in humans. Young chicks are susceptible to Salmonella and heterophils, the avian equivalent to neutrophils, are involved in control of the bacteria. To identify simultaneous host-bacterial transcriptional changes, heterophils were isolated from 6-d-old chicks, exposed to S. typhimurium for 0-1h, total eukaryotic and prokaryotic RNA isolated from the same sample, and subjected to chicken whole genome and Salmonella microarrays. Heterophils exposed to S. typhimurium upregulated 2900 and downregulated 700 genes. Changes in S. typhimurium showed 1020-25 genes were upregulated and 846-967 downregulated. Gene ontology (GO) was evaluated on differentially expressed genes. Heterophils upregulated genes associated with regulation of cell differentiation, protein transport, macromolecule localization, and heterocycle metabolic processes and downregulated actin cytoskeleton organization and biogenesis, and cellular component organization and biogenesis. GO terms for upregulated S. typhimurium genes included fatty acid biosynthesis, flagellar assembly, glutathione metabolism, and the Type III secretion system. Beginning to characterize the host-pathogen interaction and global gene expression will provide insight into potential control mechanisms for this important foodborne pathogen.
The role of c-Myc in regulating HIV-1 Tat protein induced cytokine expression and consequent effects on opportunistic infection (P4235)
Authors: Pong John , Li James , Yim Howard , Lau Allan
Published: January 2023

The human immunodeficiency virus (HIV-1) trans-activator (Tat) protein is an important viral protein that is known to contribute to the acquired immunodeficiency syndrome (AIDS) pathogenesis via cytokine dysregulation. In this study, we recognized that c-Myc regulated primary blood derived macrophage (PBMac) immune response induced by HIV-1 Tat. Treatment of PBMac with HIV-1 Tat was able to upregulate c-Myc expression in a time dependent manner. We subsequently delineated that HIV-1 Tat regulated c-Myc expression and function through the activation of dsRNA-activated protein kinase (PKR), ERK1/2 and p38 mitogen-activated protein kinase (MAPK). By knocking down the expression of c-Myc with gene specific small-interfering RNA (siRNA), we demonstrated that c-Myc may be critical for the expression of the pro-inflammatory cytokines TNF-α and IL-6. To further investigate the role of c-Myc in AIDS pathogenesis and its effects in the fight against the opportunistic microbes, Mycobacteria avium intracellulare, was used as a pathogen model. Our recent results demonstrated that HIV-1 Tat induced c-Myc to dysregulate cytokine expression, which affects the M. avium intracellular survival in PBMac. In summary, c-Myc may play a significant role in the pathogenesis of AIDS by regulating the cytokine expression by HIV-1 Tat and possibly further enhancing the infection of opportunistic pathogens in AIDS patients.
Combined radiation and thermal injury induces a non-suppressive myeloid CD11b+ Gr1+ cell population (P4025)
Authors: Linz Brandon , Brickey W. , Mendoza April , Neely Crystal , Ting Jenny , Cairns Bruce , Maile Robert
Published: January 2023

Increasing sophistication of terrorist attacks and recent nuclear energy crises have amplified the interest in the immunobiology of combined radiation and burn injury (RCI). Burn is associated with immunosuppression and increased mortality with lower doses of ionizing radiation. Following trauma, putative myeloid derived suppressor cells (MDSCs; CD11b+Gr1+) have been described. We hypothesized that RCI-MDSCs display a suppressive phenotype and RCI will result in increased susceptibility to bacterial infection. C57BL/6 female mice underwent either a 20% total body surface area full thickness contact burn or sham procedure. All mice then received a single whole body dose of 5Gy γ-radiation. Mice were infected 14 days post injury with Pseudomonas aeruginosa intratracheally. Immune cells underwent apoptosis following RCI and infection, with CD11b+Gr1(Ly6C)+ cells being the predominant surviving cell type, which we isolated and cocultured 1:1 with untouched T cells. Surprisingly, T cells incubated with CD11b+Gr1(Ly6C)+ cells from RCI mice displayed greater proliferation, increased IFN-γ and decreased IL-10 production compared to MDSC from radiation-only mice. RCI mice were able to clear the P. aeruginosa infection significantly better than radiation-only mice. These data suggest that while RCI depletes immune cells, resistant CD11b+Gr1(Ly6C)+ cells represent a target population for modulation of bacterial clearance after injury.
Timing of post-influenza bacterial superinfection with MRSA dictates the outcome of MRSA pneumonia in mice (P4027)
Authors: Rynda-Apple Agnieszka , Harmsen Ann , Richert Laura , Erickson Anfin , Harmsen Allen
Published: January 2023

Secondary bacterial pneumonias caused by S. aureus, S. pneumoniae and H. influenzae are a common occurrence following influenza infection and are responsible for significant morbidity and mortality worldwide. Others have shown in murine models that 5-8 days after influenza challenge the susceptibility to secondary bacterial infections increases; though the mechanism of this increased susceptibility is still elusive. Here, we report that 2-3 days after influenza challenge (before the onset of clinical symptoms, i.e. body weight loss) mice actually have increased resistance to secondary infection with methicillin resistant S. aureus (MRSA). Importantly, despite this early protection from MRSA, the response to bacteria early in the influenza infection results in increased viral growth and subsequently death of mice. The mechanism of this increased resistance to secondary MRSA pneumonia early after influenza is dependent on the transient presence of IL-13 and on type I IFN signaling, as mice deficient in these cytokine or cytokine receptor do not show increased resistance to secondary MRSA pneumonia 2-3 days after influenza infection, and injection of exogenous IL-13 to mice co-infected with MRSA pneumonia on day 7 increases resistance to bacteria. Thus, depending on the time of secondary infection in relation to influenza, the disease phenotype contracted by mice is greatly different, awareness of which is important for the design of rational treatments in clinics.
Role of pathogenic natural antibodies and complement in a murine model of spinal cord injury (P4051)
Authors: Qiao Fei , Zhang Lixia , Yang Xiaofeng , Hua Yunpeng , Narang Aarti , Holers Michael , Kulik Liudmila , Song Hongbin , Tomlinson Stephen
Published: January 2023

Traumatic spinal cord injury (SCI) results in complement activation and inflammation, which are implicated in a secondary injury that determines the extent of functional recovery. SCI has similar pathophysiological characteristics to ischemia reperfusion injury (IRI), and IRI in some organs is initiated by the binding of “natural” self-reactive IgM to post-ischemic neoepitopes, with the subsequent activation of complement. We demonstrated that Ab deficient Rag1-/- mice are protected from SCI, and that injury can be restored by reconstitution with a self-reactive natural IgM mAb specific for annexin A4. Following SCI, IgM bound to the spinal cord in wt mice, and anti-annexin A4 mAb bound to the spinal cord in Rag1-/- mice. Furthermore, IgM and C3d (complement activation product) co-localized after injury. We constructed a single chain antibody (scFv) derived from the anti-annexin A4 hybridoma and investigated: 1. Its ability to block the binding of pathogenic self-reactive IgM after SCI and, 2. Its utility as a targeting vehicle for delivery of a complement inhibitor after SCI (by linking the scFv to the complement inhibitor, Crry). The scFv-Crry construct, and to a lesser degree scFv alone, were protective against SCI, as measured by improved functional recovery, tissue sparing and demyelination. Data indicate an important role for self-reactive IgM in initiating complement activation after SCI, and that post-SCI neoepitopes represent therapeutic targets.
Caspase-12 inhibits obesity and insulin resistance (P4057)
Authors: Skeldon Alex , Morizot Alexandre , Kozlitina Julia , Pause Arnim , Saleh Maya
Published: January 2023

Our lab has previously identified CASPASE-12 as an inhibitor of the inflammasome, a multiprotein complex composed of a NLR, CASPASE-1, and potentially other adaptor proteins. The inflammasome has recently been identified as having a crucial role in the development of obesity and type 2 diabetes and we hypothesized that Caspase-12 may also play a role. Mice lacking Caspase-12 develop spontaneous obesity and insulin resistance. On a high fat diet (HFD), the mice develop a more severe phenotype, having increased liver triglycerides and metabolic defects including reduced respiration and activity. The adipose tissue of the Caspase-12-/- mice had increased macrophage infiltration and increased Caspase-1 processing and activation. Analysis of insulin signalling indicated that the Caspase-12-/- mice had increased insulin resistance in adipose, liver, and muscle tissue. The majority of the human population has a truncated and inactive CASPASE-12, however a proportion of people of African descent have a T125C SNP that restores the protein. To determine if Caspase-12 also had a protective effect in humans, we sequenced patients of the Dallas Heart Study for the T125C SNP. Preliminary analysis revealed a possible protective effect of a full length CASPASE-12 with respect to plasma triglycerides, however the effect was restricted to African American individuals without diabetes. No associations with body-mass index, diabetes prevalence, of fasting glucose levels were observed.
Induction of innate inflammatory responses is the major cause of liver toxicity associated with AAV-mediated RNA interference (P4007)
Authors: Tao Mi-Hua , Sun Cheng-Pu
Published: January 2023

Adeno-associated virus (AAV)-mediated RNA interference shows promise as a therapy for chronic hepatitis B virus (HBV) infection, but its low efficacy and hepatotoxicity pose major challenges. We have generated AAV vectors containing different promoters and a panel of HBV-specific short hairpin RNAs (shRNAs) to investigate factors that contribute to the efficacy and pathogenesis of AAV-mediated RNA interference. HBV transgenic mice injected with AAV vectors containing the U6 promoter produced abundant shRNAs, resulting in significant, but transient, HBV suppression and severe liver injury. AAVs containing the weaker H1 promoter did not cause liver injury, but their therapeutic efficacy was highly dependent on the sequence of the shRNA. Mice treated with the toxic U6 promoter-driven shRNA showed little change in hepatic microRNA levels, but a dramatic increase in hepatic leukocytes and inflammatory cytokines and chemokines. Hepatotoxicity was completely absent in immunodeficient mice and significantly alleviated in wild-type mice depleted of macrophages and granulocytes, suggesting that host inflammatory responses are the major cause of liver injury induced by U6 promoter-driven shRNA. Our results demonstrate that a combination of the H1 promoter and a highly potent shRNA can achieve sustained HBV suppression without inducing inflammatory side-effects.
Blocking vascular endothelial growth factor reduces granulomatous inflammation during murine mycobacterial infection (P4017)
Authors: Harding Jeffrey , Chen Yuli , Nagy Andras , Sandor Matyas
Published: January 2023

Vascular Endothelial Growth Factor is a potent pro-angiogenic factor that has broad and complex regulation over development, wound repair, and inflammation. Its importance in health and disease is underscored by its regulation over the growth and metastasis of many types of solid tumors. We have identified upregulation of VEGF protein in the granuloma during murine infection with mycobacterium. Here, using multiple approaches, we describe a previously unappreciated role of VEGF during murine infection with the Bacillus Calmette-Guerin strain of mycobacteria. We used a small peptide VEGF RTK inhibitor (SU5416), as well as employed novel transgenic mice with a hypomorphic VEGF allele (HypoVEGF Mice). We identify the cellular source of VEGF protein in the granuloma and show that, during acute infection, VEGF regulates inflammatory responses including cell accumulation and activation. Our data also show that inhibition of VEGF signaling can reduce the inflammation without limiting the host’s control of bacterial expansion. These results suggest that VEGF blockers, which have already passed phase III clinical trials in humans, could have special potential to ameliorate the fatal inflammatory responses during active tuberculosis. The finding that VEGF blockers can ameliorate granulomatous pathology could be relevant in the treatment in other diseases associated with granulomatous inflammation.
CD84 is a survival receptor for chronic lymphocytic leukemia cells (P2044)
Authors: Marom Ayelet , Binsky-Ehrenreich Inbal , Sobotta Mirko , Shvidel Lev , Berrebi Alain , Hazan-Halevy Inbal , Kay Sigi , Aloshin Anna , Sagi Irit , Goldenberg David , Leng Lin , Bucala Richard , Herishanu Yair , Haran Michal , Shachar Idit
Published: January 2023

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of CD5+ B lymphocytes in peripheral blood, lymphoid organs and bone marrow (BM). The main feature of the disease is accumulation of the malignant cells due to decreased apoptosis. CD84 belongs to the Signaling Lymphocyte Activating Molecule (SLAM) family of immunoreceptors, and has an unknown function in CLL cells. Here, we show that the expression of CD84 is significantly elevated from the early stages of the disease, and is regulated by macrophage migration inhibitory factor (MIF) and its receptor, CD74. Activation of cell surface CD84 initiates a signaling cascade that enhances CLL cell survival. Both downmodulation of CD84 expression and its immune-mediated blockade induce cell death in vitro and in vivo. In addition, analysis of samples derived from an on-going clinical trial, in which human subjects were treated with humanized anti-CD74 (milatuzumab), shows a decrease in CD84 mRNA and protein levels in milatuzumab-treated cells. This downregulation was correlated with reduction of Bcl-2 and Mcl-1 expression. Thus, our data show that overexpression of CD84 in CLL is an important survival mechanism that appears to be an early event in the pathogenesis of the disease. These findings suggest novel therapeutic strategies based on the blockade of this CD84-dependent survival pathway.
Pharmacodynamic impacts of chemotherapy and immunotherapy upon adipose tissue (P2054)
Authors: Myers Cheryl , Pollock Kevin , Bradley Judy , Gorman Jessica , Gendler Sandra , Cohen Peter
Published: January 2023

Recent guidelines by the American Society of Clinical Oncologists recommend that obese individuals receive chemotherapy based on their actual body weight (ABW) without dose reduction. Contrary to prior reports, our own data demonstrate that ABW-dosed chemotherapy or immunotherapy is as effective and well tolerated by obese mice as by lean mice. Nonetheless, obese mice develop significantly greater weight loss in a dose-dependent manner during the entire period that they receive therapy, with corresponding quantifiable reductions in visceral fat depots. This disparity is not explained by altered food intake, as both lean and obese mice experience less than a week of anorexia which does not recur after Cycle 1 of treatment. Furthermore, malabsorption is absent. These observations are relevant to multiple classes of chemo agents. Altered pharmacokinetics of chemotherapy agents in adipose tissue have been reported, yet the pharmacodynamics of obesity remain speculative. Fasting blood glucose monitoring demonstrates glucose level variances which are temporally linked to treatment, rendering it likely that chemotherapy and immunotherapy provoke adipose tissue lipolysis, or alternatively cytoreduction or immunomodulation of adipocyte progenitor cells. Understanding these effects could contribute to improved treatment regimens in the setting of obesity.
Splenectomy restores IFNγ-dependent tumoricidal activity of CD8+ T cells to promote rejection of intraocular tumors by a mechanism requiring macrophages and FasL (P2056)
Authors: McKenna Kyle , Beatty Kelly , Thorne Stephen
Published: January 2023

It is well established that splenectomy terminates ocular immune privilege to promote rejection of intraocular tumors that would otherwise grow progressively in the anterior chamber (a.c.) of the eye. However, the mechanisms of intraocular tumor rejection in splenectomized mice remain undefined. Utilizing E.G7-OVA tumors transplanted in the a.c. of C57Bl/6 mice we identified the requirement for CD8+ T cells. Interestingly, E.G7-OVA tumor growth in both splenectomized and control mice induced expansion and differentiation of OVA specific CD8+ T cells into granzyme B+ CTL that infiltrated ocular tumors in comparable numbers. As splenectomized perforin deficient mice rejected ocular tumors, these data indicated that direct lytic activity was dispensable. Rejection of ocular tumors also required macrophages and immune cell expression of IFNγR1 suggesting that splenectomy restored IFNγ production by CD8+ T cells or rescued IFNγR1 signaling in macrophages. However, IFNγ was expressed by CTL infiltrating ocular tumors of both splenectomized and control mice, and CTL-expressed IFNγ induced expression of nitric-oxide-synthase-2 in intratumoral macrophages. FasL expression was also required for ocular tumor rejection. Based on these data we propose a model in which CD8+ T cells express IFNγ to induce FasL ligand expression by intratumoral macrophages that promotes apoptosis of Fas+ tumor cells.
CpG ODN-dependent MDSC modulations contribute to the efficacy of Ad5-TRAIL/CpG ODN antitumor therapy (P2076)
Authors: James Britnie , Kucaba Tamara , Norian Lyse , Griffith Thomas
Published: January 2023

Myeloid-derived suppressor cells (MDSC) are immature, activated myeloid cells that can suppress T cell function via multiple mechanisms and dampen antitumor immunity. As with many tumor models, we found a high frequency of MDSC in the primary kidney tumor, spleen, and lung metastases in an orthotopic model of advanced renal cell carcinoma (RCC). MDSC from both primary tumor and spleen suppressed T cell proliferation in vitro. However, following treatment of the primary kidney tumor with a recombinant adenovirus encoding TRAIL (Ad-TRAIL) in combination with CpG ODN, MDSC numbers decreased at all aforementioned locations and a robust systemic CD8 T cell-mediated antitumor immune response was initiated that was ultimately responsible for clearing the primary RCC tumors and lung metastases. Subsequent analysis revealed that CpG ODN alone was able to directly modulate MDSC frequency and phenotype at the primary tumor site and metastatic lung, resulting in a more “mature” phenotype as seen by increases in CD40, CD80, and CD11c and decreases in Gr-1. Interestingly, concomitant depletion of MDSC, which alone lead to tumor regression, did not exhibit any synergistic effects with therapy, suggesting that Ad-TRAIL/CpG alone overcomes MDSC suppression. These data suggest that while CpG ODN may play many roles contributing to the antitumor immune response, one important role appears to be the modulation of the suppressive MDSC population.
Multidisciplinary approaches to enhance BRAF targeted therapy in melanoma (P2092)
Authors: Pangigadde Pradeepa Narayana , Tan Thomas , Ferrone Soldano , Karre Klas , Colucci Francesco , Carbone Ennio
Published: January 2023

40-50% of patients with melanoma are identified with BRAF mutations. About 80% of the BRAF mutations carry the V600E mutation. These patients are treated with BRAF inhibitors alone or in combination with other cell signaling pathway inhibitors. However, a relapse of the disease is observed within 6-8 months after the commencement of the therapy. Some studies hint that NK cell based therapy can be effective in controlling melanoma. In this study multidisciplinary approaches were tested to understand the mechanism of Vemurafenib resistance by a BRAFV600E melanoma. Vemurafenib is a BRAFV600E inhibitor. Vemurafenib resistant melanoma cell lines were raised from the sensitive cell lines by drug treatment till 80-90% of cells showed resistance. Both the resistant and sensitive cell lines were tested in a NK cell cytotoxicity assay in vitro by standard 51Cr-release assay. The Vemurafenib resistant cell lines were found to have a reduced sensitivity to NK cell killing. Though there was no significant difference in expression of NK cell activating ligands on the surface between the resistant and sensitive cell lines, there was a tendency of higher expression of MHC class I on Vemurafenib resistant cell lines. BRAF exon15 sequencing shows no difference between Vemurafenib resistant and sensitive cell types. Experiments are in progress to understand the mechanism(s) of the reduced sensitivity of the Vemurafenib resistant cell lines to NK cell killing.
Targeted molecular therapies for cancer: collateral damage? (P2028)
Authors: Allegrezza Michael , Rutkowski Melanie , Stephen Tom , Svoronos Nikos , Tesone Amelia , Conejo-Garcia Jose
Published: January 2023

Much attention has been given to the idea of personalized cancer treatment, where the oncogenes driving tumor growth can be identified in each patient and targeted with specific molecular therapies. Many targeted inhibitors are in clinical development after showing promise in xenograft tumor models, but their impact on anti-tumor immunity remains unknown. Because immune cells use many of the same pathways frequently mutated in cancer (MAPK, PI3K, CDK), we hypothesized that molecular inhibition of these pathways would also detrimentally affect anti-tumor immunity. We tested a panel of over 40 molecular inhibitors on TCR-induced proliferation of human T cells and found that many of these inhibited proliferation at active concentrations nearly identical to those found when tested against tumor cells. Further analysis with PI3K and MEK inhibitors showed that their effect on T cell function varied depending on the stimulation strength and presence of certain cytokines. These findings indicate that many of these molecules may be immunosuppressive, and this knowledge can help guide the development of therapies that inhibit tumor cells while maintaining protective anti-tumor immunity in order to achieve maximum therapeutic effects.
Immunotherapy of disseminated intraperitoneal pancreatic cancer with non-replicating Toxoplasma gondii (P2104)
Authors: Sanders Kiah , Fox Barbara , Bzik David
Published: January 2023

Pancreatic cancer is one of the most devastating and deadly cancers with an overall 5-year survival rate of 5%. Given existing therapies offer limited benefit, there is a strong need for innovative treatments that boost the host immune response to cancer. Toxoplasma gondii is an obligate intracellular parasite that potently stimulates the immune system and elicits CD8 T cell dependent immune responses. Utilizing an intraperitoneal dissemination model of pancreatic cancer, we hypothesize a non-replicating strain of T. gondii (CPS) can alter the tumor microenvironment and elicit strong antitumor responses. Intraperitoneal PAN02 tumors were established and treated with CPS seven days later. CPS treatment significantly increased survival and some treated mice survived long term (>1 year). Treatment efficacy was completely dependent on production of IL-12 and IFN-γ. Within the peritoneal exudate, there is a significant decrease in B cells and classical macrophages and a significant increase in neutrophils, CD4, and CD8 T cells compared to untreated tumors. Therapeutic benefit was dependent on the presence of CD8 T cells whereas CD4 T cells did not significantly contribute to the survival advantage in treated mice. CPS treatment increased the frequency of pancreatic tumor-specific IFN-γ producing CD8 T cells. Further investigation is needed to characterize innate immune mechanisms supporting tumor therapy as well as the potential induction of immune memory.
NKT cells as a novel platform for cancer immunotherapy with chimeric antigen receptors (P2038)
Authors: Heczey Andras , Liu Daofeng , Courtney Amy , Marinova Ekaterina , Wei Jie , Tian Gengwen , Yvon Eric , Hicks John , Dotti Gianpietro , Metelitsa Leonid
Published: January 2023

Transgenic expression of chimeric antigen receptors (CARs) in primary T cells is an attractive strategy for cancer immunotherapy. However, poor homing of CAR T cells to the tumor site limits their therapeutic potential. Because Vα24-invariant Natural Killer T cells (NKTs) naturally localize to solid tumors, we examined their ability to serve as a cellular carrier for anti-tumor CARs. We genetically modified primary human NKTs to express a CAR against GD2 ganglioside, which is highly expressed in tumors of neuroectodermal origin such as neuroblastoma or melanoma. CAR expression rendered NKTs highly cytotoxic against GD2-positive neuroblastoma cells without affecting their native CD1d-restricted reactivity. The comparison of CAR constructs encoding different co-stimulatory endodomains revealed a striking Th2-like or Th1-like polarization of NKTs by CARs with CD28 or CD137, respectively. CAR NKTs had potent and long-lasting anti-tumor activity in a metastatic model of neuroblastoma in humanized NOD/SCID/IL-2γ(null) mice. Furthermore, in contrast to CAR T cells from the same donor, CAR NKTs did not induce graft-versus-host disease. These results establish the potential of NKTs to serve as an effective cellular platform for anti-tumor CAR therapy.
Integrin alpha 4 differentially affect the migration of effector and regulatory T cells (P4113)
Authors: Bettelli Estelle , Glatigny Simon , Duhen Rebekka , Arbelaez Carlos , Kumari Swarnima , Oukka Mohamed , Bettelli Estelle
Published: January 2023

The integrin alpha 4 (Itga4) is expressed on immune cells and is an important molecule for the migration of T cells in various organs. Because of this propriety, a monoclonal antibody specific to Itga4 is used for the treatment of multiple sclerosis (MS). Although effective, anti-Itga4 therapy has been associated with the development of progressive multifocal leukoencephalopathy (PML), a life threatening condition, which results from a rare infection of the central nervous system (CNS) by the JC virus. Despite its use and efficacy in the clinic, it remains unclear how different cells of the adaptive immune system are affected by Itga4 deletion or neutralization. Experimental autoimmune encephalomyelitis (EAE) serves as an animal model of multiple sclerosis (MS). It results from an autoimmune attack by myelin reactive effector T helper (Th) 1 and Th17 cells and progressive demyelination. Foxp3+ regulatory T cells can control effector T cell responses but are not always effective during autoimmune disease progression. Using mice with selective deletion of Itga4 in T cells, we previously reported that Itga4 selectively inhibits the migration of Th 1 cells but not Th17 cells in the CNS. We have now established that Itga4 differentially affect the migration of effector and regulatory T cells during EAE. The implication of our findings for MS therapy will be discussed.
Pathogenesis of Systemic Lupus Erythematosus and aiCD4 T cell: new insight on autoimmunity (P4121)
Authors: Shiozawa Shunichi , Miyazaki Yumi , Tsumiyama Ken
Published: January 2023

A question in the pathogenesis of autoimmunity is why and how autoreactive lymphocytes survive beyond the firewall of immunity. According to the autoimmune disease theory, autoreactive lymphocytes derive from either a few autoreactive clones that escape from thymic negative selection or those reactivated from tolerance. Such restricted clones apparently do not represent wide varieties of TCR repertoire or autoantibody of more than 100 distinct specificities seen in SLE. We instead show that autoreactive clones newly emerge via de novo TCRα, but not TCRβ, revision at periphery from thymus-passed non-autoreactive clones. We name this novel T cell type an autoantibody-inducing CD4 T cell (aiCD4 T cell), which stimulates B cells to generate varieties of autoantibodies and also helps final differentiation of CD8 T cell into CTL via antigen cross-presentation to induce lupus tissue injuries. Our life is a continuous battle against pathogen: host dies if pathogen is too strong due to pathogen-induced tissue injury and the host also dies if defense is too strong due to defense-induced injury, because battle field is our body. To avoid exhaustive battle, our immunity evolved not to eradicate but just suppress pathogen to undersea levels. Thus, repeated exposure to antigen is not exceptional but routine. SLE necessarily emerges when host’s immune system is overstimulated by repeated exposure to pathogen to levels that surpass system’s stability-limit: self-organized criticality theory.
Analyzing the contributions of TCR affinity to the pathogenesis of experimental autoimmune encephalomyelitis (P4129)
Authors: Blanchfield J. Lori , Bettini Maria , Vignali Dario , Evavold Brian
Published: January 2023

Myelin oligodendrocyte glycoprotein (MOG) specific CD4+ T cell infiltrates from the central nervous system (CNS) have exhibited a range of affinities (low to high) for MOG peptide:MHC class II during peak EAE symptoms. Tetramer binding analyses and two-dimensional affinity assays have indicated that high affinity, MOG specific T cells comprise a rare yet significant population within the polyclonal repertoire; however, their functional contributions to the pathogenesis of chronic autoimmunity is not clear. Preliminary experiments suggested that a high precursor frequency (15 x 106) of polyclonal tetramer positive MOG specific T cells promotes early onset and greater disease severity when compared to tetramer negative T cells. Analysis of a high affinity MOG specific, CD4+ T cell (TCR Vα4β1) revealed a two dimensional affinity of 6.4x10-4 μm4 that was similar to the mean affinity (1.6x10-4 μm4) of pathogenic CD4+ T cells at peak LCMV Armstrong infection. Retrogenic models were used to assess the pathogenicity of TCR affinity in order to circumvent the limited number of endogenous high affinity T cells. The TCR Vα4β1 retrogenic mice developed spontaneous EAE even in the presence of FoxP3 positive (6%) T cells. T cells progressed though thymic selection without altering CD4 or TCR expression while maintaining their high two-dimensional affinity. Overall, MOG specific, high affinity CD4+ T cells are pathogenic and may predispose mice to a rapid progressive course of EAE.
Enhanced PI3K/Akt-mediated survival of memory Th1 cells in type-1 diabetics and healthy controls carrying the autoimmunity-associated 1858T allelic variant of PTPN22 (P4097)
Authors: Turner Michael , Habib Tania , Rieck Mary , Funk Andrew , Anderson Warren , Puri Kamal , Buckner Jane
Published: January 2023

A C-to-T polymorphism (1858T) in the PTPN22 gene alters the function of the lymphoid phosphatase Lyp and confers increased risk for autoimmunity. The PTPN22 1858T allele is associated with altered TCR signaling and expanded memory T cell pool. To determine its impact on lineage commitment and survival, naïve CD4+ T cells from healthy subjects carrying risk or non-risk alleles were differentiated in vitro. Cultures of T cells bearing PTPN22 1858T displayed similar proliferative capacity but reduced activation-induced cell death, yielding more IFNγ+ Th1 cells than non-risk controls. In vivo, PTPN22 1858T subjects mounted enhanced IFNγ responses to the pneumococcal vaccine Prevnar, but not IL-2 production or T cell proliferation. Examination of TCR-mediated signaling revealed that elevated pAkt activity and BCL2 expression were associated with the PTPN22 1858T allele and correlated with enhanced T cell survival during in vitro stimulation. This phenotype was mirrored in T cell cultures from type-1 diabetic subjects, regardless of their PTPN22 genotype, and was reversed by a PI3Kδ-specific inhibitor. Our studies indicate that the autoimmunity-associated PTPN22 1858T allele protects T cells from apoptosis via elevated PI3K/Akt signaling, leading to the accumulation of pro-inflammatory memory Th1 cells. This T-cell intrinsic defect is a general feature of T1D and inhibition of PI3Kδ offers a potential avenue for pharmacological intervention.
The TGFβ-activating integrin αvβ8 on dendritic cells regulates type 2 immunity in the intestine to promote chronic parasitic infection (P3318)
Authors: Worthington John , Klementowicz Joanna , Rahman Sayema , Czajkowska Beata , Waldmann Herman , Sparwasser Tim , Grencis Richard , Travis Mark
Published: January 2023

The cytokine TGFβ is crucial in regulating immunity in the intestine, but its role and regulation during responses to intestinal infection is not well understood. We therefore addressed how TGFβ functions and is controlled during chronic intestinal infection, using the model murine parasite Trichuris muris (the human homologue of which affects ~600 million people world-wide, causing severe health problems). We found that TGFβ signalling is induced early during infection, specifically in CD4+ T-cells. This enhanced signalling involves expression of the TGFβ-activating integrin αvβ8 by dendritic cells (DCs). Importantly, mice lacking integrin αvβ8 on DCs are completely resistant to chronic infection with T. muris, indicating an important functional role for integrin αvβ8-mediated TGFβ activation in promoting chronic infection. Protection from infection is dependent on CD4+ T-cells, but is independent of Foxp3+ Tregs as mice depleted of Foxp3+ Tregs during infection remained susceptible to infection. Instead, lack of integrin αvβ8 on DCs led to an early increase in expression of the Th2 cytokine IL-13 by CD4+ T-cells which is required for protection from infection. Our results therefore provide novel insights into how type 2 immunity is controlled in the intestine during gastrointestinal parasite infection, and may help contribute to the development of new therapies aimed at promoting expulsion of gut parasites.
MHC II restricted CD3+CD4-CD8- cells display functional characteristics of memory cells and contribute to optimal secondary immunity to Leishmania major in mice (P3337)
Authors: Uzonna Jude , Mou Zhirong , Okwor Ifeoma , Liu Dong , Jia Ping
Published: January 2023

Although it is generally believed that CD4+ T cells play important role in anti-Leishmania immunity, some studies suggest that they may be dispensable and MHC II-restricted CD3+CD4-CD8- T (double negative, DN) cells may be more important in regulating anti-Leishmania immunity. Indeed, expansion of DN cells occurs in the blood of infected patients and dogs, and in spleens of infected mice and has been proposed to contribute to immunity against Leishmania. However, concrete evidence implicating DN cells in anti-Leishmania immunity is lacking. Here, we show that following primary L. major infection, DN cells in lymphoid organs of infected mice are activated, proliferate and produce IFN-γ. Furthermore, DN cells from mice that healed their primary L. major infection display functional characteristics of anti-Leishmania memory-like cells: they rapidly and extensively proliferate and produce IFN-γ in response to L. major challenge in vitro and in vivo. Interestingly, DN cells express alpha-beta T cell receptor (αβ TCR), and are restricted by MHC class II molecules. Moreover, DN cells from healed mice, but not those from naïve mice, control parasite growth in infected macrophages in vitro. Using in vivo depletion and adoptive transfer studies, we show that DN cells from healed mice contribute to optimal secondary anti-Leishmania immunity in mice. These results demonstrate that CD3+CD4-CD8- cells play a role in both primary and secondary (memory) anti-L. major infection.
Innate amplification of CD4 T cell responses during infection: NLRs and TLRs converge (P3342)
Authors: O'Donnell Hope , Monack Denise , McSorley Stephen
Published: January 2023

During infection T cells are activated in an antigen-specific manner and can then secrete effector cytokines in response to secondary interactions with antigen-presenting cells. Effector cytokines are critical for pathogen clearance, but are tightly controlled to limit host pathology. However, mounting evidence suggests alternative non-cognate pathways can induce these effector responses. Here, we examine the amplification of Th1 CD4 T cell production of IFNγ by innate stimuli to show that this non-specific response occurs in a broad variety of infectious and stimulatory contexts and requires cooperation of both Nod-like receptor and Toll-like receptor pathways. Using knock-out and mixed bone marrow chimeric mice we show a requirement for the inflammasome, T cell intrinsic IL18 receptor and extrinsic Toll-like receptors. This suggests a model wherein infected cells undergo inflammasome activation, and upon further innate stimulation, secrete cytokines to non-specifically amplify nearby activated T cell effector responses. The mechanisms of such a pathway could lead to a better understanding of immune-mediated clearance of pathogens, as well as immune-mediated host pathology.
STAT4-dependent and -independent T helper type-2 immunity mediates protection against Pneumocystis lung infection (P3304)
Authors: Myers Riley , Dunaway Chad , Nelson Michael , Trevor Jennifer , Steele Chad
Published: January 2023

Although it is clear that the loss of CD4+ T cells is a predisposing factor for the development of Pneumocystis pneumonia, the protective mechanisms mediated by CD4+ T cells are not well understood. Augmented production of Th1-type cytokines in the lungs of IL-10-/- mice correlated with enhanced P. murina clearance, whereas neutralization of IL-17 in normal mice compromised fungal clearance, suggesting that Th1 and Th17 cells play an important role during infection. STAT4 is critical for optimal Th1 and Th17 development, therefore we investigated its role in P. murina host defense. Our data shows that STAT4 was required for Th1 and, unexpectedly, Th2 responses in the lungs of C57BL/6 (BL/6) and Balb/c mice 14 days after infection, but only Balb/c STAT4-/- mice were susceptible to P. murina infection. Th2 responses in the lungs of BL/6 STAT4-/- mice, but not Balb/c STAT4-/- mice, were intact 28 days after infection and correlated with elevated M2 macrophage polarization. Additionally, anti-P. murina class-switched antibodies were increased in BL/6 STAT4-/- mice, but not Balb/c STAT4-/- mice, as a result of hyper-Th2 responses in the draining lymph nodes. Supporting our experimental observations, we found that lower IL-4, IL-5 and IL-13 levels in plasma from HIV+ patients correlated with Pneumocystis colonization. Collectively, our data suggests that robust local and systemic Th2-mediated responses are critical for immunity to Pneumocystis.
Dendritic cells are the major antigen presenting cells in inflammatory lesions of murine mycoplasma respiratory disease and contribute to disease severity (P3306)
Authors: Simecka Jerry , Sun Xiangle , Dobbs Nicole , Bodhankar Sheetal , Jones Harlan
Published: January 2023

Mycoplasmas cause chronic respiratory diseases in animals and humans, and vaccine development has been problematic. T cell responses were shown to confer protection as well as promote immunopathology in murine mycoplasma pneumonia. Because T cells play a critical role, the role of antigen presenting cells (APC) was examined as they likely influence either an increase in disease severity or promote protective immunity. The roles of APC, such as dendritic cells (DC) and macrophages, in mycoplasma disease are currently unknown. In this study, we examined the ability pulmonary APC populations and their contribution to T cell responses during disease pathogenesis. Both macrophages and DC increased in the lungs of mice after infection. These cell populations showed different patterns of cytokine mRNA expression, supporting the idea that these cells have different impacts on immunity in response to infection. In fact, DC from the lungs of infected mice were most capable of stimulating mycoplasma-specific T helper (Th) cell responses in vitro. In vivo, DC cells were co-localized with Th cells in inflammatory lesions in the lungs of mycoplasma-infected mice. Intratracheal inoculation of mycoplasma antigen-pulsed DC resulted in increased disease severity after subsequent infection. Thus, DC appear to be the major APC population responsible for pulmonary T cell stimulation in mycoplasma-infected mice, and these DC likely contribute to responses impacting disease pathogenesis.
Laborsaving production of site-specific anti-phospho monoclonal antibodies by simultaneous screening for multiple phosphopeptides using multiplex bead assay (P3372)
Authors: Ise Tomoko , Abe Yasuhiro , Nagata Satoshi
Published: January 2023

Monoclonal antibodies (MAbs) to phosphotyrosines at specific sites of cellular proteins are critical tools to analyze the functional status of proteins. However, time consuming and laborious screening steps in current methods limits production. Here, we introduce a bead-based multiplex flow cytometry method for simultaneous evaluation of the generated MAbs for the cross-reactivity to different peptide sequences. In this proof of concept study, we chose four phosphorylation motifs from different receptors expressed on B cells (Ig-alpha, membrane IgG and IgE, and FCRL5). They were synthesized as a series of 11-mer peptides containing a tyrosine or a phosphotyrosine at the center and surrounded by unique amino acid sequences. They were conjugated with different color-coded beads which can be identified by red laser excitation in flow cytometry. Antibody binding to the peptides was measured as the level of blue laser excitable R-Phycoerythrin fluorescence. Mice immunized with phosphopeptide-KLH conjugates were used for making hybridomas. As expected, the simultaneous screening revealed unique specificities of the 10-30 MAbs generated in each fusion experiment. In particular, we obtained anti-phospho MAbs specific to Ig-alpha and membrane IgG as well as pan anti-phospho tyrosine Ab. Our results suggest that this method is suitable for production of anti-phospho MAbs that need to recognize a specific phosphopeptide among other phosphopeptides with structural similarity.
Use of antibody gene library for the isolation of specific single chain antibodies by ampicillinantigen conjugates (P3378)
Authors: Messerschmidt Katrin , Neumann-Schaal Meina , Heilmann Katja
Published: January 2023

Isolation of recombinant antibodies from antibody libraries is commonly performed by different molecular display formats including phage display and ribosome display or different cellsurface display formats. We describe a new method which allows the selection of Escherichia coli cells producing the required single chain antibody by cultivation in presence of ampicillin conjugated to the antigen of interest. The method utilizes the neutralization of the conjugate by the produced single chain antibody which is secreted to the periplasm. Therefore, a new expression system based on the pET26b vector was designed and a library was constructed. The method was successfully established first for the selection of E. coli BL21 Star (DE3) cells expressing a model single chain antibody (antifluorescein) by a simple selection assay on LB-agar plates. Using this selection assay, we could identify a new single-chain antibody binding biotin by growing E. coli BL21 Star (DE3) containing the library in presence of a biotin-ampicillin conjugate. In contrast to methods as molecular or cell surface display our selection system applies the soluble single chain antibody molecule and thereby avoids undesired effects e.g. by the phage particle or the yeast fusion protein. By selecting directly in an expression strain, production and characterization of the selected single chain antibody is possible without any further cloning or transformation steps.
Model structure of a full-length anti-IgE complexed with IgE (P3384)
Authors: Wright Jon , Sargsyan Karen , Chu Hsing Mao , Lim Carmay , Chang Tse Wen
Published: January 2023

Modeling antibody/antigen (Ab/Ag) complexes remains a challenging problem and is important for understanding how proteins associate, which in turn can aid drug design. Recently, we have implemented a method (EMAP) for producing the conformations of Ab/Ag complexes in the widely used molecular dynamics program CHARMM. Here, we apply this method to model the structure of IgE in complex with omalizumab, a humanized IgG1 antibody that has been approved in many countries for treating severe, persistent allergic asthma. First, the EMAP method was calibrated by using it to reproduce 24 nonredundant Ab/Ag complexes whose bound and unbound structures have been solved. It could yield near-native conformations for 22 of the 24 complexes, whereas other docking methods such as ZDOCK yield less near-native conformations. Having validated the EMAP method, it was used to dock full-length models of omalizumab and IgE together. The resulting IgE/omalizumab complex structure provides insights into the relative importance of the residues involved in binding. It also provides a plausible explanation for why omalizumab and IgE do not form large immune complexes.