bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of human IgM memory B cells in influenza vaccine response (P4401)
Authors: Kim Jin Hyang , Reber Adrian , Biber Renata , Cao Weiping , Chirkova Tatiana , Katz Jacqueline , Sambhara Suryaprakash
Published: January 2023

Influenza virus-specific memory B cells are critical to mount an accelerated antibody response against subsequent influenza infections. While multiple layers of humoral memory are involved in the protection against influenza, the unique role of IgM memory B cells is largely unknown. In this study, we demonstrated that human IgM memory B cells are similar to class-switched memory B cells in their basic effector functions: the ability to proliferate and to present antigens to T cells. However, ELISPOT analysis of peripheral blood mononuclear cells revealed that the frequency of influenza-specific IgM memory B cells is 10-fold higher than that of IgG memory B cells and doesn’t readily change by seasonal vaccination. Interestingly, IgM memory B cells exhibited broad cross-reactivity against contemporary seasonal as well as pandemic strains. Serum antibody analysis showed that although the level of influenza-specific IgM was approximately 20-fold less than that of IgG, the breadth of cross-reactivity of IgM-secreting cells is broader than that of IgG counterparts. Given that a recent study in mice showed that the majority of IgM memory B cells are preferentially recruited to the ensuing secondary response, we propose a model that upon vaccination, some IgM memory B cells are directly differentiated into highly cross-reactive IgM-secreting plasmablasts to confer the host early line of cross-protection, while the rest of IgM memory B cells are recruited to the secondary response.
Arming oncolytic vaccinia virus with T cell engager to improve virus therapy (P4413)
Authors: Song Xiaotong , Yu Feng , Gottschalk Stephen , Bartlett David
Published: January 2023

Oncolytic vaccinia virus (VV) therapy has shown promise in preclinical models and in clinical studies. Complete responses, however, have rarely been observed. The limitations presented by VV therapy typically have been suboptimal virus spread through the tumor and the limited induction of antigen-specific T-cell responses. Here we describe a novel strategy to overcome these limitations. We have constructed T-cell Engager Armed Vaccinia Viruses (TEA-VVs) encoding secretory bi-specific T-cell engager (single chain variable fragment, scFv) that binds both to CD3 and a tumor cell surface antigen EphA2 (EphA2-TEA-VV). The expression of EphA2-TE was demonstrated in cell culture. Also, the replication and oncolysis efficiency of EphA2-TEA-VV in cell culture were similar to that of the parental virus. However, the EphA2-TEA-VV killed EphA2-positive tumor cells A549 more effectively in the presence of human PBMC as determined by target cell lysis, compared to unmodified VV. In addition, EphA2-TEA-VV induced bystander killing of tumor cells that are not infected with virus as determined by in vitro assay. Importantly, EphA2-TEA-VV resulted in a significant decrease in tumor growth in an established A549 lung cancer model. Thus, TEA-VV represents a unique and effective strategy to improve oncolytic virus therapy by engaging T cells for cancer therapy.
TLR2 ligands modulate Fcγ receptor expression and function in monocytes (P4397)
Authors: Fatehchand Kavin , Shah Prexy , Patel Hemal , Fang Huiqing , Justiniano Steven , Mo Xiaokui , Jarjoura David , Tridandapani Susheela , Butchar Jonathan
Published: January 2023

Fcγ receptor (FcγR) clustering on monocytes/macrophages leads to phagocytosis, cytokine release and other responses designed to clear the antibody-opsonized targets. Toll-like Receptor 2 activation elicits strong proinflammatory responses, so we explored the possibility that ligands for TLR2 could indirectly strengthen FcγR activity. We treated human peripheral blood monocytes (PBM) with the TLR2 agonist Pam2CSK4 and found significantly enhanced FcγR-mediated cytokine production and phagocytosis. Further examination showed that the agonist increased the mRNA and protein levels of FcγRIIa and the common γ-subunit. Interestingly however, the inhibitory receptor FcγRIIb was also upregulated. We also found a concurrent decrease in SHIP and an increase in miR-155, which may help explain the enhanced FcγR function despite upregulated inhibitory FcγRIIb. Next, using a murine Her2/neu solid-tumor model of antibody therapy we found that Pam2CSK4 significantly enhanced antibody-mediated reduction of tumor growth, although the effects were not superadditive. Finally, we tested whether the effect was acylation-specific by treating PBM with the tri-acylated Pam3CSK4 and measuring FcγR expression and function. Results showed that Pam3CSK4 was equally effective at all dosages tested. Collectively, these findings show that TLR2 ligands can positively modulate FcγR expression and function, and hence may warrant further investigation as putative adjuvants for antitumor antibody therapy.
The two-faced T cell epitope: examining the host-microbe interface with JanusMatrix (P4399)
Authors: Gutierrez Andres , Bailey-Kellogg Chris , Moise Leonard , Terry Frances , Abdel Hady Karim , Leng Qibin , Losikoff Phyllis , Verberkmoes Nathan , Martin William , Rothman Alan , De Groot Anne
Published: January 2023

To explore the intersection of commensals, pathogens, and the human genome at the T cell epitope level, we leveraged the vast genomic sequence information available in databases of microorganisms to which humans are exposed. We developed the JanusMatrix immunoinformatic tool that identifies potentially cross-reactive T cell epitopes from both HLA binding and TCR-facing sides to allow comparison across large genome sequence databases including common human pathogens (HP), the human gut microbiome (HGM), the human genome (HG), and the human plasma proteome (HPP). Initial studies reveal different levels of HPP/HG, HGM, and HP cross-reactivity (XR) for known Treg and Teff epitopes. In Hand Foot Mouth Disease (HFMD), extensive XR with HGM seems to predict immunodominance; more limited XR with enteroviruses (e.g., polio) may protect against severe HFMD. For common Teff epitopes, HPP/HG XR is more limited than HGM XR. For Treg epitopes defined in HCV disease and for Tregitopes (De Groot et al, Blood, 2008), HPP/HG XR is more extensive. Overall, greater XR with HPP/HG compared to HGM seems to distinguish known Treg and Teff epitopes. While predicting all influences on immune responses may be impossible, the vast availability of human pathogen and commensal organism sequences now allows T cell epitope comparisons in these large datasets. Startling discoveries relevant to vaccine development and T cell response phenotype understanding are emerging as we apply this powerful technology.
Presence of lipoxin A4 during early phase of tularemia delays apoptosis of macrophages and favors exponential bacterial growth (P1354)
Authors: Singh Anju , Rahman Tabassum , Sellati Timothy
Published: January 2023

The high infectivity and virulence of Francisella tularensis (Ft) subspecies tularensis coupled with its ease of aerosolization make it an ideal bioweapon. During respiratory tularemia, caused by inhalation of Ft, we postulate that bacteria establish a principally anti-inflammatory environment and subvert host cell death programs to facilitate their unfettered replication within various cell types. Here we have explored the role of lipid mediators such as leukotrienes [i.e., lipoxin A4 (LXA4)] and prostanoids [i.e., prostaglandin E2 (PGE2)] in host inflammatory responses manifested early during infection with Ft. LXA4 has potent anti-inflammatory properties and also is involved in down-regulation of PGE2. Addition of exogenous LXA4 during in vitro infections with Ft inhibits apoptosis and also ablates the PGE2 release by macrophages, which is restored in the presence of MK 886. Our studies show that Ft facilitates its survival and exponential replication by triggering the release of LXA4, which inhibits apoptosis of macrophages and other cells within the first ~3 days of infection. We are also elucidating the course of Ft infection in 5-lipoxygenase knockout mice to observe the effects of LXA4 deficiency on the bacterial burden, histopathology and mouse survival. Targeting such lipid mediators may have therapeutic benefit by limiting bacterial growth in tularemic individuals and perhaps those infected with other highly virulent respiratory pathogens.
The histone methyltransferase Ezh2 regulates differentiation and plasticity of CD4 T helper cells. (P1334)
Authors: Tumes Damon , Onodera Atsushi , Suzuki Akane , Shinoda Kenta , Endo Yusuke , Iwamura Chiaki , Hosokawa Hiroyuki , Koseki Haruhiko , Tokoyoda Koji , Suzuki Yutaka , Nakayama Toshinori
Published: January 2023

Upon encountering antigen, naïve CD4 T cells rapidly differentiate into discrete subsets of effector T helper (Th) cells that direct immune responses to different types of infection. Polarizing cytokines induce the expression of lineage-specifying genes (Tbx21 for Th1 cells and Gata3 for Th2 cells) that control differentiation. We found that the histone H3K27 methyltransferase Ezh2 controls differentiation of Th1 and Th2 cells, and that inactivation of Ezh2 enhances plasticity of both of these subsets. Tbx21 and Gata3 experienced large changes in Ezh2 occupancy during Th differentiation, and inactivation of Ezh2 depleted H3K27-trimethylation (H3K27-Me3) at these loci. Additionally, loss of Ezh2 caused increased pathology in a mouse model of allergic asthma and progressive accumulation of memory phenotype Th2 cells in vivo. This study establishes a functional link between Ezh2 and transcriptional regulation of lineage-specifying genes in terminally differentiated peripheral T cells via H3K27-Me3. These data may therefore contribute to the development of therapies that target this pathway to enhance or repress immune responses in human diseases.
Dendritic cells in Th2 immune responses (P1313)
Authors: Ronchese Franca , Connor Lisa , Hyde Evelyn , Ochiai Sotaro , Tang Shiau Choot , Le Gros Graham
Published: January 2023

We have examined the role of dendritic cells using different models of Th2 allergic immune responses in the skin. In the first model we used the nematode parasite Nippostrongylus brasiliensis, which infects rodent hosts by penetrating through the skin. Material from labelled N. brasiliensis is taken up by migratory dendritic cells and transported to the draining lymph node where it initiates a strong Th2 immune response. We have characterized the surface phenotype of these dendritic cells in terms of lineage markers, and for expression of markers associated with Th2 immune responses. We find that these dendritic cells express markers that are compatible with a steady-state population of dermal dendritic cells. In addition, we have carried out dendritic cell transfer experiments to show that these migratory dendritic cells maintain the full capacity to prime Th2 immune responses in vivo. Additional models of Th2 immune responses, such as those initiated by the powerful allergen House Dust Mite and by application of the vitamin D analogue MC903, are currently being investigated. Our data indicate that dendritic cells can carry a sufficient set of signals to instruct the initiation of Th2 immune responses in secondary lymphoid organs.
Intraperitoneal injection of Δ-9-tetrahydrocannabinol induces local MDSCs with potent immunosuppressive properties (P1319)
Authors: Sido Jessica , Nagarkatti Prakash , Nagarkatti Mitzi
Published: January 2023

The mechanism of immune regulation by cannabinoids remains unclear. Δ-9-tetrahydrocannabinol (THC) is the most widely studied cannabinoid isolated from the Cannabis sativa plant. Our laboratory has previously shown that, in mice, THC induces myeloid derived suppressor cells (MDSCs), a heterogeneous population of immunoregulatory cells. In this study, we focused on the role of THC in MDSC expansion and generation. Intraperitoneal injection of THC induced local MDSCs which were able to suppress T-cell proliferation ex vivo, in response to mitogenic activation. THC-induced MDSCs comprised a significantly higher proportion of granulocytic MDSCs compared to MDSCs isolated from naïve bone marrow (BM). These induced MDSCs also expressed higher levels of Arginase1 than did naïve BM MDSCs. We also found that THC-induced MDSCs expressed and secreted significantly higher levels of S100A8 protein, compared to BM MDSCs, and this occurred in a Signal Transducer and Activator of Transcription (Stat) 3-dependent manner. S100A8 has been shown in tumor models to drive MDSC accumulation and activation, and may act as a chemoattractant and stimulator of local MDSCs upon THC treatment. Together these data show that in vivo THC treatment is able to induce a highly immunosuppressive granulocytic MDSC population, which may be expanded locally to restore homeostasis.
Indoleamine 2,3-dioxygenase and T-cell based anti-tumor immunotherapies targeting immune checkpoints (P2010)
Authors: Holmgaard Rikke , Zamarin Dmitriy , Munn David , Wolchok Jedd , Allison James
Published: January 2023

The CTLA-4 blocking antibody ipilimumab results in durable responses in metastatic melanoma, though therapeutic benefit has been limited to a fraction of patients. This calls for identification of resistance mechanisms and development of combinatorial strategies. We examine the inhibitory role of indoleamine 2,3-dioxygenase (IDO) on the anti-tumor efficacy of antibodies targeting immune checkpoints. We show widespread systemic induction of IDO in tumor-bearing mice. This IDO is present even after antibody treatment, demonstrating that the extensive target for IDO inhibitors is indeed present. Further, we study the anti-tumor effects of combining therapies targeting immune checkpoints with pharmacological inhibition of IDO.
Understanding the role of host innate immune responses in viral vector-mediated gene delivery and strategies to achieve sustained transgene expression (P6357)
Authors: Ankathatti Munegowda Manjunatha , Hu Jim
Published: January 2023

Stunning progress in genomic research has changed the way we address diseases. With the identification of disease causing genetic modifications, genomic research has helped us to decipher the patho-physiology of diseases and given us an extra tool to address the basic cause by correcting those genes. Only few drugs target the cause of disase, but for most of them we still need to deliver proper genes. Although many viral vectors are being used to deliver genes to target cells, most have problems related to safety and sustained expression. In our previous studies we have used HD-AD to successfully deliver cystic fibrosis correcting human-CFTR gene to mice achieving sustained expression and disease correction in CFTR KO mice. Where as in larger animals, there is an acute phase of reduction in transgene expression. Our preliminary studies suggest that, this difference may be due to difference in NK cell activity. To address this problem we have designed human cell based in-vitro evaluation assay to find out the role of NK cells and to find out effective small molecule to block NK cells during the window period of their activity. Our results show that NK cells are killing viral vector- transduced cells. We also demonstrate that this gene transduced cell killing can be inhibited by JAK and NF-kB inhibitors. These findings will help in designing better viral vector-based gene therapy to achieve sustained expression in large animals and in human studies.
Antibodies in dengue patients contain both anti-thrombotic and pro-fibrinolytic activities (P6359)
Authors: Yeh Trai-Ming
Published: January 2023

Dengue virus (DENV) infection may result in severe dengue hemorrhagic fever (DHF). However, the mechanisms that cause hemorrhage in DHF are unclear. In this study, we demonstrated that antibodies against human thrombin were increased in the sera of dengue patients but not in other virus-infected patients. To further characterize the properties of these antibodies, affinity-purified anti-thrombin antibodies (ATA) were collected from DENV patients’ sera by thrombin and protein A/L affinity columns. We found human ATA bound not only to DENV, human thrombin, but also cross-reacted to human plasminogen (Plg). In addition, human ATA could inhibit not only thrombin activity but also enhance Plg activation in vitro. Taken together, these results suggest that antibodies induced by DENV, which contain both anti-thrombotic and pro-fibrinolytic activities, may contribute to the development of hemorrhage in DHF.
Cooperative functions of MHC II, HLA-DM, and cathepsins enhance the selection of immunodominant epitopes (P5008)
Authors: Kim Ae , Hartman Isamu , Poore Bradley , Boronina Tatiana , Lanar David , Krzych Urszula , Cole Robert , Sadegh-Nasseri Scheherazade
Published: January 2023

We developed a minimalist cell-free antigen-processing system for MHC class II that can identify physiologically selected T cell epitopes from antigens. The system utilizes purified proteins: HLA-DR1, cathepsin B, H, S, and HLA-DM. It was designed to mimic the specialized compartment for antigen processing with known defined molecular composition so that it can be a useful tool for elucidating steps involved in antigen processing and understanding the mechanisms of epitope selection. We found that prior digestion of antigens even for a few minutes eliminated the possibility of their immunodominant epitope capture by DR1. However, if those antigens were captured by DR1 first in the presence of DM, dominant epitopes were successfully selected. We observed simultaneously binding of DR1 and DR4 to denatured proteins detected by the biacore method. In another case, pre-digestion of antigen did not destroy the dominant epitope even though it showed sensitivity to DM mediated dissociation. We saw that this epitope got selected because it was less susceptible to enzymatic digestions and it was able to rebind to DR1 even in the presence of DM. Therefore, In general, most peptides generated from proteins do not get a chance to bind MHC II for presentation because they are sensitive to both DM and enzymatic digestions. Selection of immunodominant epitope is the results of collaborative reactions of DR1, DM, and cathepsins by increasing abundance of epitope capture by MHC II.
Triggering receptor expressed on myeloid cells 2 induces obesity by promoting adipogenesis (P3107)
Authors: Park Min , Choi Harim , Kang Hyung-Sik
Published: January 2023

The role of TREM2 in adipogenesis and obesity has not yet been defined. Here, we report that TREM2-transgenic (TG) mice were much more obese than wild-type mice after feeding with a high-fat diet (HFD), independent of the quantity of food intake. These HFD-fed TREM2 TG mice manifested adipocyte hypertrophy, glucose and insulin resistance and hepatic steatosis. The expression of adipogenic regulator genes, such as peroxisome proliferator-activated receptor γ, CCAAT/enhancer-binding protein α, fatty-acid synthase and CD36, was markedly increased in the epididymal white adipose tissue (EWAT) of the HFD-fed TG mice. In addition, the EWAT of the HFD-fed TG mice exhibited decreased Wnt10b expression and increased GSK-3β-mediated β-catenin phosphorylation. In contrast, the blockade of TREM2 signaling using TREM2-Ig resulted in the inhibition of adipocyte differentiation in vitro and a reduction in body weight in vivo by downregulating the expression of adipogenic regulators. Taken together, these results suggested that TREM2 induces obesity by promoting adipogenesis via the upregulation of adipogenic regulators in conjunction with the Wnt10b/β-catenin signaling pathway.
Essential role for Toll-like receptor 9 in the pathogenesis of liver inflammation in a murine model of Crohn’s disease-like ileitis (P3147)
Authors: Omenetti Sara , Brogi Marco , Garg Rekha , Goodman Wendy , Klinman Dennis , Pinzani Massimo , Pizarro Theresa
Published: January 2023

Liver inflammation, a common extraintestinal manifestation of inflammatory bowel disease (IBD), was reported to unexpectedly precede ileitis onset in SAMP1/YitFc (SAMP) mice, a spontaneous model of IBD. We hypothesized that since SAMP possess an inherent epithelial barrier defect that can facilitate bacterial translocation, ultimately into the liver, toll-like receptor (TLR) activation of resident immune cells may occur, leading to liver inflammation. To test this, SAMP X MyD88KO mice were generated and liver inflammation assessed. TLRs were measured by qPCR and TLR9+ cells identified by FACS in isolated non-parenchymal liver cells (NPLs). Cytokines were measured by ELISA from NPLs +/- a TLR9 agonist, and SAMP treated with a TLR9 agonist or antagonist evaluated. Deletion of MyD88 resulted in complete amelioration of SAMP liver inflammation as early as 4, and up to 40, wks vs age-matched WT SAMP (both p<0.001). TLR9 mRNA levels were highest for TLR expression in SAMP vs AKR (parental) control livers (↑2.4-fold, p<0.001) and TLR9 expressed in both NPL F4/80+ MΦs and CD4+ T cells, with specific TLR9 activation modulating TNF, IL-12, and IL-10 (p<0.05). Finally, administration of a TLR9 agonist exacerbated (p<0.001), while a TLR9 antagonist ameliorated, SAMP liver inflammation (vs vehicle, p<0.05). These data indicate a pivotal role for TLR9 in the pathogenesis of liver inflammation in ileitis-prone SAMP mice and suggest its importance in hepatic involvement in patients with IBD.
Clinical and immunological profile of patients with corneal ulcers due to microbial keratitis (P3157)
Authors: Jimenez-Martinez Maria C , Santacruz Concepcion , Estrada-Garcia I Iris , Perez-Tapia Mayra , Garfias Yonathan
Published: January 2023

Aim: To describe clinical and immunological characteristics of human microbial keratitis. Methods. 28 patients with bacterial and fungal keratitis were included. Characteristics of ocular lesions such as size of corneal ulcer (epithelial defect), infiltration, hypopyon, were considered; immunological evaluation included determination of IL-1b, IL-6, IL-8, IL-10, IL-12 and TNF-a by cytometric bead arrays in samples obtained from infected and non-infected eyes; frequency of CD4, CD8, CD19 and CD56 lymphocytes were also determined on peripheral blood mononuclear cells (PMBC). Results. Non-significant differences were observed in area of epithelial defect, stromal infiltration, nor hypopyon, when compared; however, we observed a characteristic immunological profile apparently related to keratitis origin: IL-8 >IL-6 in bacterial keratitis; IL-8>IL-6 with increased frequency of circulating CD3-CD56+ cells in gram-negative keratitis; and IL-8=IL-6 >IL-1b in fungal keratitis. Conclusions. This study is the first in evaluate clinical and immunological characteristics of patients with corneal ulcers, despite we did not observe a clinical profile clearly related with the type of keratitis, characterization of immunological status could aid to understand immune pathophysiological mechanisms underlying each type of keratitis. IL-6 and IL-8 could be a target for therapy in severe microbial keratitis
The effect of an autoimmune-associated allelic variant of protein tyrosine phosphatase nonreceptor 22 on B cell tolerance in a murine model. (P4066)
Authors: Metzler Genita , Rawlings David
Published: January 2023

The C1858T allelic variant of tyrosine phosphatase PTPN22 (resulting in an R620W change of protein product, LYP) is strongly associated with multiple autoimmune diseases. Contradictory findings suggest the LYP-R620W variant exhibits either gain- or loss-of-function activity in antigen-receptor signaling. To model this risk allele in mice, we generated knock-in mice expressing the analogous R619W mutation in the murine ortholog, PEP. While the variant protein exhibits normal stability, its expression significantly alters lymphocyte function and aged knock-in mice exhibit effector T and, transitional, germinal center and age-related B cell expansion, autoantibodies and systemic autoimmunity. Further, PEP-R619W impacts B cell selection and, strikingly, B lineage-restricted variant expression is sufficient to promote autoimmunity. In the current project, we initiated a detailed study of the impact of variant expression on B cell tolerance mechanisms. Our studies were designed to test the hypothesis that while negative selection is intact, peripheral B cells expressing the variant exhibit increased positive selection, culminating in a skewed, autoreactive naïve B cell repertoire that promotes autoimmune pathogenesis. Preliminary data utilizing single cell BCR cloning and deep sequencing technologies to assess autoreactivity and V(D)J family usage in naïve B cell subsets, as well as characterization of negative and peripheral selection using in vivo models, will be presented.
Linear synthetic peptides induce anti-HER-2 antibody responses with Trastuzumab- (Herceptin) like biological activity (P4308)
Authors: Showalter Loral , Czerniecki Brian , Koski Gary
Published: January 2023

The monoclonal antibody Trastuzumab, which recognizes a conformation-dependent epitope on the extracellular domain of HER-2, has revolutionized the treatment of HER-2pos breast cancers. High costs and short half-life of Trastuzumab spurs the search for ways to induce therapeutic anti-HER-2 antibodies in vivo. Linear T cell epitopes have already been discovered for HER-2, and these have shown promise in vaccine studies where strong cell-mediated responses have been generated. Identifying conformation-independent B cell epitopes would allow the construction of linear T-B vaccine constructs that would generate both cellular and humoral anti-HER-2 responses with potential anti-cancer activity. We produced a synthetic peptide library consisting of 62 overlapping 20-mers spanning the HER-2 extracellular domain. These were used to vaccinate Balb/c mice. Resulting antisera were screened for reactivity against peptide and recombinant extracellular domain protein via ELISA, and whole native protein by immuno-fluorescent microscopy and FACS analysis. Sera reactive by all these criteria were compared with Trastuzumab for ability to disrupt HER-2 and HER-2/HER-3 dimer signaling with and without heregulin. We identified linear peptides that induced antibodies capable of blocking AKT as well as HER-3 phosphorylation in breast cancer lines similar to Trastuzumab. This suggests that a linear epitope/vaccination approach may be feasible for generating effective anti-HER-2 antibody responses.
Expression of CXCR5 and its natural ligand CXCL13 in breast cancer (P6278)
Authors: Singh Rajesh , Gupta Pranav , Grizzle William , Singh Shailesh , Lillard, Jr. James
Published: January 2023

Despite recent advancements in understanding of breast cancer (BrCa) molecular features and their impact on clinical behavior, BrCa systemic therapy decision-making is still guided by a constellation of clinico-pathological features, which are derived from histo-pathological analysis of primary BrCa tissues. Chemokines and their corresponding receptors play a crucial role in metastasis and organ specific homing of cancer cells in different cancers including BrCa. CXCL13 is a homeostatic chemokine acts through its cognate receptor, CXCR5. Here, we have provided the evidence that CXCR5 and CXCL13 are significantly elevated in both BrCa cell lines (MCF-7 and MDA-MB-231) and clinical samples. Flow cytometric analysis of BrCa cell lines (MCF-7 and MDA-MB-231) show significantly higher expression of CXCR5 in comparison to normal epithelial cells (MCF-10A). This finding was further confirmed at mRNA expression level by real-time PCR analysis. To determine the clinical significance of CXCR5 expressed by BrCa cell lines, breast cancer tissue microarray was stained for CXCR5 and CXCL13. Aperio scan scope scanning system was used to numerically analyze immuno-histochemical staining of CXCR5 and CXCL13. Our results show significantly higher expression of CXCR5 and CXCL13 (p < 0.001) in cancerous tissues compared to normal breast tissues. These data demonstrate both biological and clinical significance of CXCR5 and CXCL13 expression that may contribute to BrCa metastasis.
NK inhibitory receptor Ly49G2 recognition of MHC I Dk in efficient virus control (P6058)
Authors: Coats Ebony , Xie Xuefang , Deshmukh Umesh , Brown Michael
Published: January 2023

NK cells bearing inhibitory receptor Ly49G2 preferentially respond to and efficiently control murine cytomegalovirus (MCMV) in MA/My and C57L mice with Dk. Thus the role of Ly49G2 and Dk in virus control is under study. One hypothesis to explain the necessity of Ly49G2 and Dk is “missing self” recognition—release of inhibition due to downregulation of MHC I—by Ly49G2 of Dk during infection. To confirm Ly49G2 binding to Dk, we generated reporter cells expressing chimeric Ly49G2. We also performed licensing and cytotoxicity assays to assess Ly49G2+ NK cell licensing and inhibition of Dk, thus confirming Dk as the cognate ligand of Ly49G2. Next we examined Ly49G2 recognition of Dk on infected cells. Recent data has shown MCMV immunoevasin gp34 bound to MHC I stabilizes interactions between inhibitory receptors and MHC I, including the BALB allele of Ly49G2. However as BALB.K mice with Dk have 3-fold higher MCMV titers than MA/My or C57L mice with Dk, we examined the role of gp34 in Ly49G2c57l recognition of Dk. We show preferential association of gp34 to Dk, not Kk. Further, binding assays also confirm gp34 stabilization of Ly49G2mamy binding to Dk on infected cells. As cis Dk expression with Ly49G2 may impact trans binding, we are examining the role of cis binding of Dk. Additionally, signaling may be impacted by gp34, causing “missing self” recognition by Ly49G2. As such, Ly49G2 phosphorylation is being assessed, as well as phosphorylation of downstream inhibitory molecules.
Out of sequence “signal 3” suppresses CD8 T cell proliferation (P6040)
Authors: Urban Stina , Welsh Raymond
Published: January 2023

Type 1 interferons (IFN), the first cytokines to be discovered, have been shown to regulate many aspects of the immune system. Type 1 IFN can act directly on CD8 T cells as a “signal 3” cytokine, which augments proliferation and differentiation of T cells into effectors in response to antigen. Exposure of CD8 T cells to IFN-inducers at the same time as antigen had stimulatory effects, whereas pre-exposure to IFN-inducers was inhibitory, indicating that the timing of IFN exposure was of essence. Pre-exposure of CD8 T cells to the type 1 IFN inducer and TLR agonist poly(I:C) had transient inhibitory effects on CD8 T cell proliferation. CD8 T cells lacking type 1 IFN receptors were resistant to the suppressive effects of poly(I:C), indicating a direct involvement of IFN. Poly(I:C)-pretreated CD8 T cells were refractory to further IFNβ stimulation, as measured by phosphorylation of STAT molecules, including STAT1, 3, 4, and 5. Reduced phospho-STAT levels were not due to a decrease in total STAT protein. Poly(I:C)-pretreated CD8 T cells phosphorylated STAT molecules in response to other cytokines, including IL-6 and IL-15. A decrease in type 1 IFN receptors and increased expression of SOCS1 likely contribute to the unresponsiveness to IFNβ stimulation. These data suggest that IFN-pretreated CD8 T cells are unable to get the positive effects that type 1 IFN provides as a “signal 3” cytokine, and this may partially explain why vaccines function poorly in virus-infected individuals.
Early IL-17 production by intrahepatic T cells is important for adaptive immune responses in viral hepatitis (P6042)
Authors: Jie Zuliang , Hou Lifei , Liang Yuejin , Sun Jiaren
Published: January 2023

This study was conducted to examine the interactions among the innate and adaptive immune components of the liver parenchyma during acute viral hepatitis. Mice were i.v. infected with a recombinant adenovirus. We found a transient, but significant, accumulation of IL-17-producing cells in the liver within the 24 h of infection. In vivo neutralization of IL-17 alleviated the liver injury. Further investigations showed that IL-17 neutralization halted the intrahepatic accumulation of CTL and Th1 cells. Additionally, the serum ALT levels, liver injury and the functions of intrahepatic CTL and Th1 cells were significantly suppressed in IL-17R-/- mice compared to the controls. A majority of the IL-17-producing cells in the liver were γδ T cells. Notably, intrahepatic IL-17+ γδ T cells, but not the IFN-γ+ ones, preferentially expressed IL-7Rα (CD127) on their surface, which coincided with an elevation of hepatocyte-derived IL-7 at 12 h post-infection. IL-7Rα blockade in vivo severely impeded the expansion of IL-17-producing cells following viral infection. In vitro, IL-7 synergized with IL-23 and stimulated IL-17 production from γδ T cells in response to TCRγδ stimulation. Collectively, this study showed that the IL-7/IL-17 cascade played a critical role in initiating the adaptive anti-viral responses in viral hepatitis. The cross talk among hepatocytes, innate and adaptive immune cells may be critical to mount the successful adaptive anti-viral immune response in the liver.
Differential T cell responses to lingering viral antigen following the resolution of infection. (P6044)
Authors: Misumi Ichiro , Whitton Lindsay , Whitmire Jason
Published: January 2023

The contraction phase after acute virus infection is a poorly understood period when virus-specific T cells decline in number but continue to evolve functionally until achieving memory cell homeostasis. After lymphocytic choriomeningitis virus (LCMV) infection, virus-specific CD4+ T cells decline in number gradually over a 1-2 month period before establishing stable levels of memory. We find that naïve virus-specific CD4+ T cells continue to divide in response to residual LCMV antigen for several weeks after the eradication of infectious virus. The cell division of CD4+ T cells during this time is slower than that seen during the acute phase of infection when there is live replicating virus, but faster than the homeostatic cell division observed in long-term immune mice. The viral antigen driving this cell-division has a half-life of about 40 days. The presentation of this antigen does not depend on B cells, FcγRII, or antigen-antibody complexes. In contrast to CD4+ T cells, CD8+ T cells halt cell division within days after acute LCMV infection is resolved, indicating that CD8+ T cell responses are tightly linked to endogenous processing of de novo synthesized virus protein and the cross-presentation of residual viral antigen to CD8+ T cells is inefficient during the contraction phase. Our results indicate that the longevity of residual viral antigen governs the duration of the helper T cell contraction phase, serving to sustain T cell numbers and influence the memory pool.
Recirculation and retention contribute to a population of mature Tregs in the thymus. (P1153)
Authors: Yang EnJun , Zou Tao , Zhang Shirley , Hall Maclean , Kambayashi Taku
Published: January 2023

Regulatory T cells (Tregs) are a subset of T cells that are important in maintaining immunological tolerance. After final development in the thymus, T cells are thought to quickly leave the thymus to populate peripheral secondary lymphoid organs. However, in contrast to conventional T cells, we hereby report that a large fraction of Tregs reside in the thymus for a prolonged period of time. Based on studies with RAG2.GFP reporter mice and bone marrow chimeras, we found that newly developed Tregs represent <50% of the thymic Treg population in adult mice. The fraction of Tregs occupied by newly developed Tregs decreases further with age. We next tested whether the pool of mature Tregs (not newly developed) in the thymus was formed by recirculation of Tregs from the periphery and/or by Tregs that never leave the thymus. Adoptive transfer studies suggested that Tregs can recirculate back to the thymus. In addition, we found that Tregs are retained in the thymus for a prolonged period of time, as Tregs of donor origin were detected in donor thymi 6 weeks after thymic transplantation. Finally, using a combination of adoptive transfer and bone marrow transplantation, we estimate that compared to recirculation, thymic retention of Tregs contributes twice as much to the establishment of the mature Treg pool in the thymus. The thymic resident Treg population may affect the development of new Tregs in order to control antigen specificity or numbers of newly developing Tregs.
TCR signaling by conventional T cells is required for peripheral maintenance of Foxp3+ regulatory T cells (P1155)
Authors: Leichner Theresa , Satake Atsushi , Kambayashi Taku
Published: January 2023

Regulatory T cells (Tregs) control inappropriate conventional T cell (Tconv) activation. Treg maintenance is highly dependent on their IL-2-mediated proliferation in the periphery. Since Tconvs but not Tregs produce IL-2, IL-2 could serve as a gauge for Tregs to adjust their numbers accordingly to the size of the Tconv pool in the periphery. As the production of IL-2 by Tconvs is dependent on TCR signaling, we hypothesized that TCR signaling by Tconvs in response to self MHC class II (MHC-II) molecules is required for Treg maintenance. Indeed, when TCR signaling was specifically ablated in Tconvs but not in Tregs, an abrupt drop in Treg numbers was observed. Moreover, Treg numbers could not be maintained when T cells were adoptively transferred into MHC-II-deficient bone marrow chimeric mice, where MHC-II molecules were missing on radiosensitive antigen presenting cells. The drop in Treg numbers was associated with Tconv activation and the development of severe dermatitis, gut pathology, and weight loss. The decrease in Treg numbers and the onset of this disease was delayed by the injection of IL-2. These results suggest that IL-2 produced through TCR signaling by Tconvs in response to self MHC-II molecules is critical for the maintenance of Tregs and for subsequent prevention of a widespread inflammatory syndrome. This points to a potential role of positive selection in producing Tconvs bearing TCRs with adequate affinity to self MHC-II complexes in Treg maintenance.
Differential selection of Th1 effector and memory pools depends on the qualitative nature of TCR-driven CD4+ T cell activation (P1175)
Authors: Williams Matthew , Kim Chulwoo
Published: January 2023

Not all clones that participate in the effector Th1 response to intracellular viral or bacterial infection can give rise to memory T cells. In this study, we generated a mouse with a fixed TCRα chain of known specificity in order to characterize the evolution of effector and memory TCRβ repertoires following LCMV or Listeria infection by deep sequencing. The repertoire diversity of antigen-specific Th1 cells significantly decreased in the transition from effector to memory, and hierarchical cluster analysis revealed that the TCR repertoires of memory T cells, regardless of infectious model, were more similar to each other than to the effector Th1 populations from which they arose. We further generated a large panel of TCRs derived from our sequencing results that were cloned into retroviral expression vectors and used to generate TCR “retrogenic” bone marrow chimeras. Individual clonal T cell populations harvested from these mice were analyzed in adoptive transfer experiments to allow for their longitudinal analysis following infection. We found that the individual memory potential of each clone did not correspond to overall TCR avidity, as measured in tetramer equilibrium binding assays, but to the acquisition of high functional avidity and slow off-rates, as determined by tetramer decay. Our findings support a model in which differential selection of the Th1 effector and memory pools depends at least in part on the qualitative nature of TCR-driven CD4+ T cell activation.
The role of Foxp3 in augmented homeostatic proliferation of Treg (P1187)
Authors: Lin Yan , Camirand Geoffrey , Rothstein David
Published: January 2023

Natural CD4+Foxp3+ Tregs (nTreg) exhibit a high rate of constitutive homeostatic proliferation (HP) compared to CD4+Foxp3- conventional T (Tconv) cells (~50% vs. <10% proliferate over 10d, respectively, as determined by transfer of CFSE-labeled CD4 subsets into replete recipients). nTreg from Filig mutant mice (~10% wt Foxp3 level), exhibited low HP (<10%). Transducing Filig nTreg with Foxp3 retrovirus (rv), restored high HP (40%) vs. vector control (12% HP). However, when Foxp3 rv was expressed in Tconv cells, the resulting induced Tregs (iTregs) exhibited similar HP to Tconv cells transduced with vector control (17% vs. 16%, respectively). Thus, Foxp3 is necessary but not sufficient for high HP. Next, we examined the role of Foxp3 in HP of endogenously selected T cells (using EdU labeling). Filig bone marrow transduced with Foxp3 rv vs. control rv was transplanted into irradiated congenic hosts. Surprisingly, in this setting, Tconv cells transduced to constitutively express Foxp3 (iTregs), underwent high HP (32%) compared to control rv (or nontransduced) Foxp3- counterparts (12% HP). Thus, forced Foxp3 expression during thymic ontogeny augments HP capacity, not seen when Foxp3 is acquired by mature Tconv in the periphery. These findings provide new insight into the regulation of HP in Treg, the role of Foxp3 expression in Treg homeostasis, and highlight additional distinctions between induced and natural Treg.
CLEC16A: A novel C-type lectin domain-containing protein pertaining to systemic lupus erythematosus (P4092)
Authors: Tam Rachel CY , Yan Sheng , Lau Chak Sing , Chan Vera SF
Published: January 2023

Several independent genome wide association studies have shown associations of single nucleotide polymorphisms within the C-type lectin domain family 16 member A gene, CLEC16A, with various autoimmune diseases, including systemic lupus erythematosus (SLE). However, the expression and function of this novel C-type lectin domain-containing protein is still largely unknown. This study aims to identify the expressed forms of CLEC16A in peripheral mononuclear cells (PBMCs) and to relate its expression level with SLE disease activity. Two expressed isoforms, isoforms 1 and 2, of CLEC16A have been cloned and sequenced from PBMCs from healthy Chinese, each bearing differences in sequence when compared to the reference sequences derived from Caucasians. In isoform 1, exon 5 was consistently absent in Chinese PBMCs. However, in isoform 2, the stretch of 48-bp in-frame deletion within exon 11 in the reference sequence was found present in Chinese PBMCs. The mRNA expression levels of both isoforms were found significantly lower in SLE patients than in normal individuals. Yet, their expression levels did not correlate with disease activity as measured by SLE disease activity index. The molecular and cellular functions of CLEC16A are currently under investigation, which should shed light on the importance of this protein in SLE pathogenesis.
Adoptive transfers reveal dual roles in the protection against arthritis induction in the DACP rat (P4096)
Authors: Norin Ulrika , Rintisch Carola , Tuncel Jonatan , Bäckdahl Liselotte , Ekman Diana , Holmdahl Rikard
Published: January 2023

The DA rat is a commonly used inbred rat strain in inflammation research due to its susceptibility to numerous experimentally induced models of autoimmunity. We have isolated a spontaneous mutation that abrogates induction of a panel of inflammatory disorders in the DA rat and mapped it to a 2,5Mb region on chromosome 9. This new subset of the DA rat was called DA completely protected, DACP. To elucidate if the protection seen in the DACP rat is conveyed during the induction or the effector phase we utilized the Pristane-induced arthritis transfer model in a reciprocal fashion. Our results showed that cells from the DACP rat did not transfer arthritis to susceptible DA rats whereas cells from the susceptible DA rat caused a mild arthritis in DACP recipients. Looking at the cytokine profile of these cells we saw that cells from the DACP rat produced twice the amount of IL-10 compared to cells from the DA rat and that the production of RANTES was lower in the DACP rat. Differences in levels of IL-10 and RANTES could be one possible explanation for the protection seen in the DACP rat however this remains to be shown. In summary, the DACP rat was unable to develop disease upon arthritis induction but could develop a mild arthritis when receiving arthritogenic cells from the DA rat. Our findings suggest a possible dual role of the spontaneous mutation in the induction phase and the effector phase of arthritis in the DACP rat.
MicroRNA-mediated modulations in hyperactive plasmacytoid dendritic cells in systemic lupus erythematosus (P4072)
Authors: Yan Sheng , Tam Rachel CY , Chan Vera SF , Lau Chak Sing
Published: January 2023

Systemic lupus erythematosus (SLE) patients are typically presented by their increased serum type I interferon (IFN) activities. Plasmacytoid dendritic cells (pDCs), the most potent type I IFN-producing cells, are found to be hyperactive in SLE. Using the New Zealand Black/White F1 lupus mouse model, we sought for the regulatory mechanism of IFN production by pDCs in SLE. Mice with lupus symptoms such as high titers of serum anti-nuclear antibodies and persistent proteinuria were compared with the pre-symptomatic ones. Upon toll-like receptor (TLR) 7 and TLR9 stimulations, the up-regulations of co-stimulatory markers CD40, CD86 and MHC class II were significantly heightened in bone-marrow-derived pDCs from the symptomatic mice. Furthermore, the expression profiles of over 700 microRNA (miRNAs) in pDCs upon TLR7 activation were analyzed by low-density arrays. Among those, miRNA-155 was the most highly induced and its induction was consistently higher in pDCs from the symptomatic mice. It was previously found in human studies that miRNA-155 regulates type I IFN production by pDCs and itself inversely regulated by the autocrine/paracrine IFN dynamics. Current investigations pursue on the correlation between up-regulated miRNA-155 induction and aberrant pDC functions in SLE using miRNA mimics and inhibitors. Together, our study should reveal miRNA-mediated modulations in pDC abnormalities in SLE.
Immune and ribosomal gene expression associate with relapse in ADAMTS13-deficient thrombotic thrombocytopenic purpura (P4086)
Authors: Edgar Contessa , Terrell Deirdra , Vesely Sara , Dozmorov Igor , Wren Jonathoan , Frank Mark , Merrill Joan , Kremer Hovinga Johanna , James Judith , George James , Farris Amy
Published: January 2023

40% of patients who survive acute episodes of thrombotic thrombocytopenic purpura (TTP) associated with severe ADAMTS13 deficiency will relapse. Risk factors for relapse other than severe ADAMTS13 deficiency are unknown. ADAMTS13 autoantibodies, TTP episodes following infection or type I interferon treatment and reported ensuing systemic lupus erythematosus in some patients suggest immune dysregulation. This study asked whether ADAMTS13-deficient TTP patients in remission have peripheral blood transcriptional markers that associate with relapse history. Peripheral blood from 38 patients with acquired autoimmune ADAMTS13-deficient TTP in remission was examined for autoantibodies, type I interferon activity and global gene expression. A subset of patients (1.4-26.6 year follow-up; median 7.6 years) displayed a peripheral blood type I interferon gene signature that associated with elevated serum type I interferon activity and autoantibodies to RNA-binding proteins but not relapse. A second subset of patients (4.1-26.4 year follow-up; median 11.2 years) exhibited a peripheral blood gene signature composed of ribosomal transcripts that associated with relapse history. Patients who had relapsed also expressed higher natural killer cell, T lymphocyte and HLA transcript levels. The immune transcripts positively correlated with episode number but not with time since last episode. These data link a ribosomal/immune gene signature with relapse in acquired, ADAMTS13-deficient TTP.