bluuebunny/crossref_metadata_2025 huggingface dataset:
Autophagy regulates mycobacterial uptake by modulation of scavenger receptors (P4215)
Authors: Bonilla Diana , Bhattacharya Abhisek , Sha Youbao , Xu Yi , Xiang Qian , Gillard Baiba , Pownall Henry , Khan Arshad , Jagannath Chinnaswamy , Komatsu Masaaki , Eissa Tony
Published: January 2023

Autophagy and phagocytosis are highly conserved cellular functions involved in innate immunity. However, the nature of the interactions between these two critical processes remains essentially unknown. We evaluated the role of autophagy in regulating phagocytosis by measuring phagocytic activity in autophagy-deficient macrophages obtained from a myeloid-specific autophagy-related gene 7 knockout mice Atg7-/-. Importantly, in addition to increased mycobacterial growth, Atg7-/- macrophages exhibited higher bacterial uptake, when infected with Mycobacterium tuberculosis or M. tuberculosis var. bovis BCG. In addition, Atg7-/- mice, infected intranasally with BCG, showed increased bacterial loads and exacerbated inflammatory responses in lungs, compared to Atg7+/+ cells. Atg7-/- macrophages had increased surface expression of two class A scavenger receptors: macrophage receptor with collagenous structure MARCO and scavenger receptor-A. These findings were confirmed in Atg3-/-, Atg5-/- and Atg7-/- mouse embryonic fibroblasts. The increased surface expression of scavenger receptors was caused by increased activity of the transcription factor NF-E2-related factor 2 due to accumulation of p62 in autophagy-deficient macrophages. These new insights increase our understanding of host-pathogen relationship and suggest that therapeutic strategies against pathogens should be designed to include modulation of both phagocytosis and autophagy.
Adenosine binding A2B adenosine receptors is an essential danger signal for the initiation and development of the type 2 immune response to the intestinal parasite Heligmosomoides polygyrus (P4041)
Authors: Wu Wenhui , Patel Nirav , Mishra Pankaj , Hasko George , Gause William
Published: January 2023

A2B adenosine receptor (A2BAR) signaling modulates lymphocyte function in vitro and can control proinflammatory immune responses. To examine whether A2B receptors contribute to the in vivo protective type 2 immune response to helminthes, A2BAR-/- mice were infected with H. polygyrus (Hp). Worm burden and egg production were increased while serum IgE and IgG1 were reduced in A2BAR-/- mice compared to WT mice at day 14 after secondary Hp inoculation. Worms recovered from A2BAR-/- mice at day 11 after secondary inoculation exhibited higher protein and ATP levels than similarly inoculated WT mice, suggesting impaired host protective responses. Th2 cytokine expression and M2 macrophage markers were also inhibited in small intestines and in mesenteric lymph nodes (MLN) in A2BAR-/- mice during early luminal phase after secondary inoculation. After primary inoculation, increases in IL-33, and ILC2 cell activation and expression of IL-5 and IL-13 were inhibited in A2BAR-/- mice. IL-33 administration to Hp inoculated A2BAR-/- mice rescued ILC2 activation and cytokine expression. These studies indicate specific binding of adenosine to A2BAR, likely as a result of tissue damage, triggers the production of the alarmin IL-33 which then initiates the type 2 immune response. These results identify for the first time an essential danger associated molecular pattern required for the development of the helminth-induced type 2 immune response.
Withaferin A is effective in inhibiting the survival of B cell lymphomas and chronic lymphocytic leukemias (P2052)
Authors: Gachuki Beth , Oben Karine , Munniappan Latha , Alhakeem Sara , Rangnekar Vivek , Muthusamy Raj , Byrd John , Gupta Ramesh , Bondada Subbarao
Published: January 2023

Withaferin A is a withanolide from the ayurvedic medicinal plant, Withania Somnifera that has been shown to be a major contributor to the medicinal value of extracts from this plant. Withanolides, oxidation products of steroids, are known to have growth inhibitory effects on certain insect larvae. Withaferin A is the first withanolide to be isolated from the Aswagandha plant. It has been shown to have anticancer activity in breast and lung cancer models but its effects on hematological malignancies have not been studied in detail. Here we examined the effect of highly purified Withaferin A on the survival of diffuse large B cell lymphoma cell lines. Withaferin A strongly inhibited the survival of Ramos, Sudhl6, LY10 and murine A20luc and BKS2 B cell lymphoma cell lines, mainly by affecting cell cycle progression. It also reduced the survival of chronic lymphocytic leukemia cells (CLL) from the Emu-Tcl1 transgenic mice. We found that Withaferin A inhibited activation of Akt rapidly but of Src family kinase after 24 hours of incubation and also reduced the levels of pro-survival protein Bcl-x , in the murine CLL cells Withaferin A also reduced the levels of the pro-survival oncogene Tcl1 as well as the pro-apoptotic protein Par-4. We are currently investigating the mechanisms that underlie the opposite effects (reduction of both pro-survival and anti-survival proteins) and their relation to the anti-lymphoma/leukemia effects of Withaferin A.
Therapeutic potential in cancer: using adenoviral vaccine vector co-expressing tumorantigen and interleukin-2 (P2100)
Authors: Jensen Benjamin , Steffensen Maria , Christensen Jan , Holst Peter , Thomsen Allan
Published: January 2023

Cytokines role in the adaptive immune system has drawn significantly attention in recent years. An important finding is that T-cell quality and T-cell numbers tend to decline in cancer patients, partly as a consequence of inadequate amounts of the cytokine interleukin-2 (IL-2), due to a general tumor environmental down regulation of the T-cell activating molecules CD80/86. Thus, co-administration of cytokine(s), in combination with an antigen of interest, possesses the potential to modulate the immune response in a favorable direction. As a consequence, we have modified an adenoviral vaccine vector; designed it to co-express the glycoprotein (GP) from lymphocytic choriomeningitis virus (LCMV), alongside IL-2, in two disperse reading frames. Both components are exchangeable and expressed autonomously. Remarkably, adenoviral vaccine vectors have been postulated to be among the most potent and promising candidates for production of viral vaccines. The results exhibit increased tumor protection in wild type (WT) mice and impressive T-cell responses in CD80/86 knockout mice, which are otherwise known to have a diminished immune response, but most importantly, the described vaccine candidate enables CD80/86 knockout mice to restore tumor protection, undertake levels equivalent to WT mice. A concern regarding the use of IL-2 expressing vectors is the impact on the generation of long-term memory. However, we did not obtain any evidence pointing towards reduced CD8 T-cell memory.
α-Galactosylceramide analogues in cancer immunotherapeutics (P2102)
Authors: Sharma Shalu , Veerapen Natacha , Bhowruth Veemal , Zhang Lianjun , Donda Alena , Besra Gurdyal , Porcelli Steven
Published: January 2023

Natural Killer T cells (NKT) initiate potent anti-tumor responses upon activation by glycolipid antigens presented by CD1d, a non-classical MHC class I-like antigen presenting molecule. Anti-tumor responses are induced following NKT cell activation through their direct production of cytokines such as IFN-γ, and through a variety of indirect effects including dendritic cell maturation, the activation of NK cells and the cross-priming of tumor-specific CD8+ T cells. The glycolipid α-galactosylceramide (α-GC) is a CD1d ligand and potent NKT cell activator that enhances anti-tumor immune responses and decreases metastases in vivo. However, the response is short-lived, and causes liver toxicity and long term anergy of NKT cells. Here we report a novel class of chemically modified α-GCs, designated GCBs, that demonstrate unique binding properties to both mouse and human CD1d and enhanced NKT cell stimulation in vitro, as compared to previously described NKT cell activating ligands. A range of analyses have been carried out to characterize the structure and stability of GCB:CD1d complexes, revealing unique features that distinguish them from previously analyzed α-GC:CD1d complexes. Soluble GCB:CD1d complexes are currently being tested in vivo in mouse models for their ability to drive the sustained activation of NKT cells, which may provide a superior approach for development of improved cancer immunotherapies.
Reducing Fli1 levels in the MRL/lpr lupus prone mouse model impacts T cell activation and cytokine production (P4115)
Authors: Nowling Tamara , Richard Erin , Zhang John , Siskind Leah
Published: January 2023

The Ets factor Fli1 is implicated as a key modulator of lupus disease expression. Over-expressing Fli1 in healthy mice, results in the development of an autoimmune kidney disease similar to that observed in lupus. Lowering the global levels of Fli1 in two lupus mouse models significantly improved kidney disease and prolonged survival. The mechanisms by which Fli1 exerts this protective effect are not fully understood. We demonstrate that MRL/lpr lupus prone T cells expressing 50% less Fli1 (Fli1+/-) produce significantly less IL-4 and IL-13 and exhibit significantly reduced TCR-specific activation. Neuraminidase 1 (Neu1), a mediator of glycosphingolipid metabolism is required for TCR-specific activation and IL-4 production in T cells. Our results demonstrate that MRL/lpr Fli1+/- T cells express significantly reduced levels of Neu1 message and that Fli1 can regulate Neu1 promoter activity in T cells. Furthermore, MRL/lpr Fli1+/- T cells express significantly reduced levels of the glycosphingolipids lactosylceramide and glucosylceramide. Together, these results suggest that Fli1 may mediate lupus disease progression by modulating activation and cytokine expression of lupus T cells in part by regulating Neu1 expression and glycosphingolipid metabolism.
Affinity of T cells for chromogranin A remain constant in type I diabetic NOD mice. (P4119)
Authors: Hood Jennifer , Evavold Brian
Published: January 2023

In type 1 diabetes numerous autoantigens have been identified in the non-obese mouse model including insulin B 9-23, chromogranin A (ChgA29-42), and glutamic acid decarboxylase 65 (GAD65) to list a few, but the kinetics of antigen specificity during the progression from insulitis to overt diabetes is not known. A T cell clone, BDC2.5, isolated from a diabetic NOD was recently identified as being reactive to ChgA. The two-dimensional (2D) micropipette adhesion frequency assay was able to determine that BDC2.5 TCR transgenic cells specifically recognize ChgA29-42, albeit with 100-fold lower affinity compared to the highly stimulatory mimotope. As this TCR was originally derived from a diabetic NOD, we wanted to see if this highly sensitive assay could detect ChgA29-42 specific cells in the pancreatic lymph nodes (PLN) of diabetic NODs. ChgA29-42 specific CD4 T cells were detected in the PLN at a high frequency and their 2D effective affinity was not statistically different from the BDC2.5 affinity for ChgA29-42. Tracking both the frequency and affinity of antigen specific cells isolated from normoglycemic to overtly diabetic female NOD mice, we have determined that the frequency of antigen specific cells present in the PLN but not the overall affinity range changes for ChgA29-42 between these time points. Thus the micropipette adhesion frequency assay allows one to track the frequency and affinity of antigen specific T cells during the progression to autoimmune disease.
Characterization of the cytokine and transcription factor profile of CD4+ T cells from conditional LATY136F mice (P4133)
Authors: Swan Greg , Sullivan Sarah , Zhang Weiguo
Published: January 2023

LAT is an adaptor protein that is required for TCR signaling and T-cell activation. LATY136F mice have a point mutation at tyrosine 136 of LAT (Latm/m) whichprevents LAT from interacting with PLC-γ1 resulting in defective calcium flux and ERK activation upon TCR stimulation. Our lab created conditional LATY136F mice (LATf/m) in which the floxed allele can be deleted upon tamoxifen treatment. T cells from these mice undergo normal thymic development yet when thewildtype LAT allele is deleted in the periphery, develop a lymphoproliferative disease similar to LATY136F mice. Using these mice we were able to characterize transcription factor and cytokine production over the course of disease development in isolation from defective thymic development. We observed similar symptoms of disease to LATY136F mice includingsplenomegly and lymphadenopathy. Unlike CD4 cells from LATm/m mice, we observed, using immunostainingand qRT-PCR, elevated expression of Th1 cytokines and transcription factors during disease development that then declined by week 5. We also observed a stable population of cells that expressed both Th1 and Th2 cytokines. Finally we observed a transient decline in the pro-survival transcription factor BCL-2 and a steady decline of the pro-survival factor MCL-1. From this data we conclude that disease development in theperiphery of LATf/m mice is dynamic and differs from disease development in LATm/m mice.
Lateral distribution of the T cell receptor and cholesterol detected by high-resolution secondary ion mass spectrometry (P3370)
Authors: Wang Feng , Lozano Monica , Lillemeier Björn , Boxer Steven , Davis Mark
Published: January 2023

The engagement of the T cell antigen receptor (TCR) by a specific peptide-MHC ligand initiates a trans-membrane signaling that activates the T cell. An important event in T cell signaling is the recruitment of signaling proteins to the cell membrane and the assembly of a multi-protein (and lipid) signaling complex. Although it has been suggested that lipid/cholesterol composition plays an important role in the regulation of surface signaling complexes, direct visual evidence on lipid/cholesterol participation in the formation of these complexes is still lacking. Here, we characterize the clustering of TCR within T cell and model membranes by secondary ion mass spectrometry (NanoSIMS) with a spatial resolution of 50 nanometers or better. Quantitative information about the chemical composition of membrane complexes can be obtained through the use of orthogonal isotopic labeling of each signaling molecule. Analysis of nanoSIMS images shows the accumulation of TCRs into 60-150 nm clusters/islands within the membrane of an activating T cell and we further attempt to reveal the composition and lateral distribution of TCR and other membrane components, including cholesterol, within model membranes. Because these membrane structures may be shared with many other cell types, we believe the insights we gain in this system will be generalizable to other examples of cell surface receptor signaling.
Massively parallel phenotyping and clonotyping of single T cells (P3380)
Authors: Johnson David , Conant Carolyn , Withey Gary , Löhr Andrea , Hsu Andro , Meyer Everett
Published: January 2023

Flow cytometry captures T cell functionality but not individual T cell receptor clonal sequences. DNA sequencing characterizes the T cell clonotype repertoire, but lacks functional information. We report a method, T cell receptor-Effector Linkage Sequencing (TELS), which uses single-cell microfluidics and next-generation sequencing (NGS) to determine clonotype and phenotype for millions of T cells simultaneously. Single T cells are encapsulated in picoliter droplets. Overlap extension reverse transcriptase PCR amplifies T cell receptor beta (TCRb) and immune effector transcripts separately in early cycles. In later cycles, a linker DNA sequence drives synthesis of fusion products containing both TCRb and effector amplicons. After sequencing the fused products, we process the NGS data to determine clonotype-specific effector expression for each clonotype in the sample. We characterized single-cell expression levels for TCRb, Fas ligand (FasL), and interleukin 2 (IL-2) in four mouse T cell lines using qPCR. Using these cell lines spiked at 0.1% in murine peripheral blood, we produced TELS libraries with a set of multiplexed primers targeting the full TCRb repertoire, FasL, and IL-2. Across >50 experiments, we accurately quantified (p<2.2x10-16) T cell spike-in clonotype and phenotype. TELS could be used routinely for applications where knowing both T cell clonotype and function are helpful for discovery, such as transplantation, vaccine development, and cancer.
Techniques for thymic decellularization (P3392)
Authors: Falcon Jessica
Published: January 2023

Decellularization protocols for both whole organs and tissue parts have been developed and successful for many organs but not for the thymus. The decellularization process maintains the integrity of the organ by preserving the components of the extracellular matrix, including proteins such as collagen and fibronectin. This allows for an acellular, three dimensional biological scaffold of the desired organ which can then be repopulated with cells. Routine decellularization techniques published for other tissue such as the heart and the lung were inadequate for the thymus and ablated the extracellular matrix. Novel techniques were discovered. The aim of this project is to apply the idea of decellularization to the thymus, using thymic tissue procured from murine, porcine, and human models. Thymic tissue has been decellularized using two methods; SDS (sodium dodecyl sulfate) based protocols and by Deoxycholic Acid/Triton-X mixture protocols, and examined for remnant cells and extracellular proteins through histological staining and SEM imaging. We have confirmed complete decelluarization with murine thymic tissue and are advancing to porcine and human samples. We have identified varying decelluarization techniques that provide multiple degrees of integrity of the extra cellular matrix. These techniques may be used in tissue engineering strategies to repopulate a thymic construct for thymic reconstitution.
Fast and easy access to the overall B cell repertoire using the Blood B Booster® system (P3360)
Authors: Razanajaona-Doll Diane , Bertaux Ségolène , Clear Olivier , Laurant Stéphanie , Pin Jean-Jacques
Published: January 2023

Overall B repertoire analysis usually requires extensive molecular analysis of immunoglobulin genes. Determination of serum levels of specific antibodies by ELISA is restricted to “non steady-state” cases such as following vaccination, bacterial or viral infections. Dendritics has developed the Blood B Booster® system, a powerful B cell immortalization tool based on stimulation through CD40 and immortalization with Epstein-Barr virus. Briefly, the Blood B Booster® system is intended to generate B cell oligoclones representative of the overall B cell population. Here, using blood sample isolated from a healthy donor, we demonstrated that natural autoantibodies against various cytokines are detected at variable frequencies by ELISA in the supernatants of B cell oligoclones after 28 days of culture. Furthermore, we showed that Blood B Booster® is a suitable readout system to evaluate the effects of a given monoclonal antibody on B cell growth and/or Ig secretion. Finally, extension of the Blood B Booster® system to animal species allowed us to perform comparative studies between canine and human humoral responses against a candidate vaccine. In conclusion, analysis of the natural autoantibody repertoire, of the antibody responses against therapeutic agents (candidate vaccine, therapeutic mAbs) and human/animal comparative studies will be facilitated by the Blood B Booster® system which is shown here as an essential tool giving a fast and easy access to the overall B repertoire.
Simultaneous analysis of multiple fluorescent proteins and fluorochromes by a novel spectral flow cytometer (P3364)
Authors: Tomura Michio , Futamura Koji , Furuki Motohiro
Published: January 2023

We have been revealing immune system based of spatiotemporal regulation of immune cells in the entire body by using photoconvertible fluorescent protein (FP), Kaede and KikGR mice. Flow cytometric analysis of green and red signals of non-photoconverted and photoconverted Kaede or KikGR expressing cells with multiple fluorochrome-conjugated mAbs have been difficult and unable to increase color panels, because of the fluorescence signal overlap between FPs and fluorochromes. We have newly developed a spectral flow cytometer (FCM) based on a novel measurement principle. Unlike a polychromatic FCM, the spectral FCM with 32 channel linear array PMT detects the fluorescence derived from every fluorescent probe. The data of acquired spectra is analyzed with a unique algorithm. We detected spectral changes of KikGR color from green to red during photoconversion. We separated EGFP and Venus. One of the feature advantages of this instrument is that it can recognize spectral shape of each fluorescent probes and we could separate EGFP and FITC, their peaks of wavelength are almost same and only spectral shape of GFP is slightly broader than FITC. Finally, we successfully separated 11-colors including kikGR-green and -red simultaneously. Taken together, spectral FCM allow us to detection of FPs with multicolor fluorochromes, and thus, the system which uses KikGR mice with spectral FCM is a powerful tool to investigate spatiotemporal regulation of immune cells in the entire body.
Chemerin aggravates dextran sulfate sodium-induced colitis by impairing the protective innate responses (P3274)
Authors: He Rui , Lin Yuli , Yang Xuguang , Zou Linlin , Li Bingji , Yue Wenjie
Published: January 2023

Chemerin is characterized by a chemoattractant protein present in various inflammatory sites. Several studies have suggested that chemerin plays an anti-inflammatory role in acute tissue inflammation by inhibiting the recruitment of inflammatory cells. It has been recently reported that chemerin is elevated in patients with inflammatory bowel disease (IBD), however, the role of chemerin in IBD remains unknown. In the present study, we examined a role for chemerin in the development DSS-induced experimental colitis. Chemerin expression was up-regulated on days 5 and 8 after continuous DSS exposure for 5 days. Administration of exogenous chemerin aggravated the severity of DSS-induced colitis, which was characterized by more severe clinical disease scores, extensive epithelial damage and significantly increased expression of inflammatory cytokines in the colon on days 5 and 8. The exacerbated colitis was associated with decreased early colonic infiltration of neutrophils and decreased colonic expression of M2 macrophage-associated genes. In vitro study further demonstrated that chemerin negatively regulated IL-4-induced M2 macrophage polarization from the peritoneal macrophages. Our results suggest that chemerin plays an inflammatory role in DSS-induced colitis by impairing the protective innate responses and the targeting of chemerin for anti-inflammatory treatment should be cautious.
Spontaneous and persistent activation of NLRP3/NALP3 inflammasome plays a critical pathological role in colitis of IL-10 deficient (IL-10-/-) mice (P3240)
Authors: Li Xuhang , Liu Ling , Ye Mei , Yang Jianbo , Joosse Maria , Marohn Michael
Published: January 2023

IL-10-/- mice spontaneously develop chronic colitis. Our study was to evaluate the role and regulation of NLRP3 inflammasome in colitis of IL-10-/- mice and Crohn’s disease (CD). Two major NLRP3 complex proteins, NLRP3 and ASC, were upregulated in colonic mucosa of both IL-10-/- mice and CD. Importantly, NLRP3 inflammasome in IL-10-/- mice was activated prior to the onset of colitis; it progressively increased as disease worsened, peaking as macroscopic disease emerged. NOD2, a major IBD susceptible gene product, was upregulated only at a later stage of colitis when NLRP3 inflammasome was fully activated. The NLRP3 complex, including NLRP3, ASC and caspase-1, was demonstrated in vivo in colonic mucosa and increased in IL-10-/- mice, using 3 independent approaches (density fractionation, crosslinking, and co-IP). NLRP3 inflammasome activation occurred spontaneously in macrophages of IL-10-/- but not WT mice, and was effectively inhibited by IL-10. Glyburide, an inflammasome inhibitor, suppressed NLRP3 inflammasome expression/activation in IL-10-/- mice, resulting in not only alleviation of preexisting colitis, but also prevention of colitis development when given before disease onset. Thus, spontaneous and persistent activation of NLRP3 inflammasome, which is negatively regulated by IL-10, plays a major pathogenic role in colitis, it functions as a disease initiator whereas NOD2 may act as an accelerator. Inhibitors of the NLRP3 inflammasome may have utility in IBD therapy.
TRAF5 negatively regulates Toll-like receptor signaling (P1226)
Authors: Buchta Claire , Bishop Gail
Published: January 2023

Dysregulation in Toll-like receptor (TLR) signaling has been implicated in various inflammatory and autoimmune disorders. The cytoplasmic adaptor proteins TNF receptor associated factor (TRAF)3 and TRAF6 are known to mediate TLR signaling. Data from our lab suggest for the first time that another family member, TRAF5, is a negative regulator of TLR signaling. B lymphocytes, macrophages, and dendritic cells from TRAF5-/- mice have enhanced early signaling pathways including increased phosphorylation of the kinases ERK1/2, JNK, and Akt . They also produce more cytokines after TLR ligation. Consistent with these results, overexpression of TRAF5 in B cells inhibits TLR-induced cytokine production. Pursuing the mechanism of TRAF5-mediated regulation, we discovered that TRAF5 negatively regulates the transcription and protein levels of TRAF6. Additionally, TRAF5 associates in a complex with TRAF3, TRAF6, and the TLR adaptor protein MyD88 after TLR stimulation in B cells. Results suggest multiple mechanisms by which TRAF5 negatively regulates TLR signaling. TRAF5 deficiency has been reported to accelerate atherogenesis in mice. Elucidating the role of TRAF5 in TLR signaling may thus provide a new target for therapies against a variety of inflammatory conditions.
CD4+FoxP3+ regulatory T cell-mediated suppression of dendritic cells (P1049)
Authors: Liu Sang , Joly Anne-Laure , Mailer Reiner , Andersson John
Published: January 2023

CD4+FoxP3+ regulatory T cells are a subset of T cells that play central role in regulating inflammatory responses during many different immunological diseases. However the mechanisms of CD4+FoxP3+ regulatory T cells-mediated suppression remain controversial and poorly understood. Several studies have shown that CD4+FoxP3+ regulatory T cells can interact with and suppress a large number of cell types including CD4+ T cells and antigen presenting cells. For instance in vitro studies have shown that CD4+FoxP3+ regulatory T cells can inhibit cytokine production especially IL-2 production on CD4+ T cells hence the inhibition of CD4+ T cell proliferation; CD4+FoxP3+ regulatory T cells can also confer infectious tolerance by converting conventional T cells into functional FoxP3+ T cells in a cell contact TGF-β-dependent manner.In addition, CD4+FoxP3+ regulatory T cells suppress dendritic cell maturation and function by down-regulating the expression of co-stimulatory molecules CD80/CD86 via the interaction with CTLA-4.In our study, we have observed that CD4+FoxP3+ regulatory T cells may also modulate dendritic cell function via the PD-1:PD-L pathway. Dendritic cells that have been previously cultured with CD4+FoxP3+ regulatory T cells have decreased ability to support CD4+CD25- T cell proliferation and they can also induce higher level of FoxP3 expression in CD4+CD25- T cells.
Potentiation of regulatory T cell stability and function via a neuropilin-1:semaphorin-4a axis (P1057)
Authors: Delgoffe Greg , Woo Seng , Turnis Meghan , Gravano David , Guy Cliff , Bettini Matthew , Finkelstein David , Vogel Peter , Bonnevier Jody , Workman Creg , Vignali Dario
Published: January 2023

Regulatory T cells represent a critical immunoregulatory component of the immune system. The signals that maintain Treg stability and potentiate their function remain obscure. Here we show that the immune cell surface ligand semaphorin-4a on conventional murine and human T cells and dendritic cells interacts with the Treg-restricted receptor neuropilin-1 to potentiate Treg function and enhance survival. Mice with a Treg-restricted deletion of Nrp1 exhibit extremely limited tumor-induced tolerance, and thus substantial resistance to certain tumors, yet do not develop any autoimmune or inflammatory manifestations. Sema4a or Nrp1 blockade also had therapeutic efficacy against pre-existing tumors. In addition, Nrp1-deficient Treg cells fail to suppress inflammatory colitis. Upon Sema4a ligation, Nrp1 was recruited to the immunological synapse and repressed Akt activity via PTEN, which facilitated Foxo nuclear translocation. This induced a transcriptional program that promoted Treg stability, survival and function while repressing the induction of lineage-specific transcription factors. Thus, Nrp1 ligation enforces Treg stability and function in highly inflammatory sites but is dispensable for the maintenance of immune homeostasis, highlighting Nrp1 as a potential immunotherapeutic target in cancer.
Virus-specific Tregs are terminally differentiated upon peripheral activation and dependent on thymic output after both primary and secondary infection (P1035)
Authors: Liu Jie , Kim Sung-Han , Cao Shirley , Peppers Gretchen , Graham Barney
Published: January 2023

Regulatory CD4 T cells (Tregs) are defined as FoxP3+ cells and are categorized as natural (nTreg) or induced (iTreg). iTregs can be further classified as pathogen or epitope specific (pTreg). pTregs can share epitope specificity with effector cells (Teffs) and expand in response to infection. Although pTregs reduce infection-induced immunopathology, over-expansion and persistence can have a negative impact on pathogen clearance. Studying CD4 T cell responses to respiratory syncytial virus (RSV) infection, we found that the pTreg response had different dynamics than Teffs, which promoted efficient RSV clearance with minimal immunopathology and effectively established adaptive memory responses. The IA(b)M209-specific pTreg response was subdominant to IA(b)M209-specific Teffs. It peaked early to suppress responses in lymphoid organs after primary infection, but had limited regulation on CD8 T cell responses at the site of infection. The IA(b)M209-specific pTreg response contracted when the specific Teff responses peaked, and was diminished after secondary infection when an adequate adaptive Teff responses were established. The dynamics and differentiation pattern of the IA(b)M209-specific pTreg response was TCR clonotype-specific and enriched for recent thymic immigrants even on secondary infection, which suggests that pTreg responses are recalibrated with each infection to provide an optimal balance of Teff-mediated virus clearance and immunoregulation to limit tissue damage.
Cellular sentinels for heat shock protein-peptide complexes in vivo. (P4270)
Authors: Messmer Michelle , Binder Robert
Published: January 2023

Heat shock proteins (HSPs), such as gp96, harvested from cancer cells elicit potent anti-tumor immunity in mice and in patients with cancer. HSPs in the extracellular environment, released by cells or introduced through vaccination, are taken up by antigen presenting cells of the immune system, which allows for cross-presentation of the HSP-chaperoned peptides to T-cells. The goal of this study is to identify the subset(s) of cells that sample HSPs in vivo prior to initiating T cell responses. By vaccinating mice with fluorescent-labeled gp96, cells in the draining lymph node (dLN) that engage extracellular HSPs have been explored in a time and dose dependent manner. Our results show that CD4+CD11b+ cells are the predominant cells that internalized the HSP in the subcapsular region of the dLN while CD11c+CD8+ cells, which have been shown by other groups to be the major cross-priming dendritic cells, did not significantly internalize the HSP at any time points. Other CD11c+ cells did not appreciably internalize HSP until 24+ hours after. Internalization corresponded to higher CD91 expression by the CD4+CD11b+ cell subset. The functional capabilities of the CD4+CD11b+ and CD11c+ cells in T cell priming are examined. These studies illustrate how HSPs act to alert the immune system to cellular damage and will be useful for optimizing HSP-based vaccination regimes by identifying possible cellular targets.
Elucidating the role of bovine heavy- and light-chain variable domains in polyspecific antigen-binding (P6104)
Authors: Pasman Yfke , Kaushik Azad
Published: January 2023

The bovine antibody repertoire is generated from a limited germline sequence divergence as well as combinatorial diversity. Generation of an exceptionally long CDR3H (up to 61 amino acids) in the heavy chain variable region (VH) provides an additional mechanism to generate antibody diversity, not found in other species to date. These long CDR3H loops, present in polyspecific IgM, originate from VDJ recombination encoded by a specific VH gene, unusually long single DH-gene (capable of encoding up to 51 codons) and insertion of 15-18 base long conserved short nucleotide sequences (CSNS) specifically at VH-DH junction. In cattle, it is thought that antigen-binding is mainly a function of the variable-region of the heavy chain where light chain provides only structural support. To test this hypothesis, single chain variable fragment (scFv) and single domains (Fd) from polyspecific IgM were constructed. Both purified scFv as well as FdVH showed polyspecific binding to structurally dissimilar antigens in an ELISA. Whether scFv and FdVH bind to multiple epitopes on an antigen or if they recognize the same epitope can now be determined. The structural-functional complexities of these antibody fragments and the role of the heavy- and light-chains in antigen-binding will be discussed. [Supported by NSERC Canada]
Reactivity of bovine WC1+ γδ T cells to viral dsRNA (P6107)
Authors: Toka Felix , Krukowska Ewelina , Golde William
Published: January 2023

Bovine γδ T cells have properties of both innate and adaptive immune cells. However, detailed studies of how bovine γδ T cells execute their functions as innate or adaptive immune cells are lacking. In addition, their precise role in viral infections of cattle is not known. We have examined the reactivity of bovine γδ T cells to viral dsRNA isolated from bovine respiratory syncytial virus. Viral dsRNA was added to sorted peripheral blood WC1+ γδ T cells or directly delivered into the cells with DOTAP. Cell surface expression of CD80, CD335, MHC I and II, and synthesis of TNFα and IFNγ were investigated following stimulation with viral dsRNA. Addition of viral dsRNA to the culture of γδ T cells does not lead to changes in expression of CD335, MHC I and II but induces increased expression of CD80, although not statistically significant compared to non-stimulated cells. Direct delivery of viral dsRNA into WC1+ γδ T cells by DOTAP induced a twofold increase in expression of CD335 and MHC I with smaller increases in expression of MHC II and CD80. Although WC1+ γδ T cells synthesized low levels of TNFα and IFNγ in the presence of viral dsRNA or not, they significantly increased synthesis of IFNγ upon direct delivery of viral dsRNA into the cells. Results suggest that viral dsRNA may induce expression of molecules that promote innate function of γδ T cells and are discussed in context of pathways for enhancing bovine γδ T cell function against viral infections.
Polymorphisms and tissue expression of the Feline Leukocyte Antigen class I loci FLA I-E, -H and -K (P6086)
Authors: Hess Paul , Holmes Jennifer , Holmer Savannah , Gojanovich Gregory , Ross Peter
Published: January 2023

The domestic cat is a popular model of human retroviral diseases, but surprisingly, knowledge of the MHC class I complex (FLA I) in this species is limited. Only a few full-length coding sequences with uncertain locus assignments are known, and allelic variability, genotypic frequency and expression patterns remain undetermined. Of 19 class I genes, 3 loci - FLA I-E, -H and -K - are predicted to encode classical molecules. Our objective was to confirm their presumptive class Ia status by investigating polymorphisms and tissue expression of these genes. To accomplish this task, we developed a PCR-based, locus-specific, genotyping protocol, and then amplified 33 different FLA I-E, -H, and -K alleles from the gDNA of 12 cats of various breeds. We identified, for the first time, allele genotypes across all three loci in a feline species. We also amplified alleles from cDNAs from eight different tissues using an FLA I multi-loci-permissive primer set; only FLA I-E, -H, and -K transcripts were detected, and all three were widely expressed. The putative proteins encoded by our FLA I-E, -H, and -K alleles are highly variable and fit the class Ia model of homologous domains. Previously reported full-length FLA I sequences were confirmed, as were the predictions of classical status from the feline genome; FLA I-E, -H, and -K meet two major criteria of class Ia genes. These data lay the important groundwork for studying epitope-specific cytotoxic T-lymphocyte responses in the cat.
Fowlpox-based Survivin vaccination for malignant mesothelioma therapy (P2117)
Authors: Bertino Pietro , Panigada Maddalena , Soprana Elisa , Rose Aaron , Gaudino Giovanni , Carbone Michele , Siccardi Antonio , Hoffmann Peter
Published: January 2023

Survivin is an attractive candidate for cancer immunotherapy since it is abundantly expressed in most human cancers and absent in normal adult tissues. Malignant mesothelioma (MM) is a deadly cancer associated with asbestos exposure with no successful therapies currently available. We evaluated a Survivin-based vaccine as therapeutic tool for malignant mesothelioma in different MM mouse models. BALB/c mice, injected subcutaneously or intraperitoneally with murine fiber-induced MM tumor cells, were vaccinated with recombinant Fowlpox virus replicons encoding Survivin. Vaccination generated immune responses in both models, which led to delayed tumor growth and improved survival. Flow cytometric and immunofluorescent analyses of tumors from vaccinated mice showed CD8+ T cells infiltration, and real-time PCR demonstrated increased mRNA and protein levels of immunostimulatory cytokines. ELISPOT and intracellular cytokine staining analyses of Survivin peptide pulsed spleen cells from vaccinated mice confirmed antigen-specific, interferon-γ-producing CD8+ T cell responses. In addition pentamer-based flow cytometry showed vaccination generated Survivin specific CD8+ T cells. Importantly, vaccination did not affect fertility or induce autoimmune abnormalities in mice. Our results demonstrate that vaccination with recombinant Fowlpox expressing Survivin improves targeting of aggressive MM cells by the immune system and may form the basis for promising clinical applications.
Tumorigenesis of NIH3T3 cells induced by the exogenously over-expression of the novel tumor antigen OVA66 (P2121)
Authors: Ge Hailiang , Rao Wei , Xie Guohua , Li Weiyi , Xi Yibin , Wang Ying
Published: January 2023

The tumor associated antigen OVA66 which is firstly defined by serological analysis of human ovary cancer of recombinant cDNA expression library, has been demonstrated to be highly expressed in the majority of malignant tumors. We constructed a eukaryotic expression vector pFlag-OVA66 to establish an OVA66 stably over-expressed mouse fibroblast NIH3T3 cell line. The OVA66 over-expressed NIH3T3 cells exhibited several significantly malignant changes, the S and G2/M phage was markedly increased whereas the G1/G0 phage was decreased in the total cell cycle, indicating that the over-expression of OVA66 is able to promote cell cycling and proliferation. MTT and colony formation assay validated that OVA66 promotes the cell growth and colony formation in vitro. NIH3T3 cells with the over-expression of tumor antigen OVA66 displayed more resistance to the cell apoptosis induced by 5-FU. In vivo assay of the tumor xenograft studies in nude mice revealed the OVA66 over-expressed NIH3T3 cells were capable of forming tumors in the nude mice compared to the NIH3T3-mock cells with low expression of OVA66. Analysis of the phosphorylation of AKT and ERK1/2 stimulated with serum indicated a hyper activation of ERK1/2 MAPK and PI3K/AKT pathway in OVA66 stably over-expressed NIH3T3 cells. The results suggesting that OVA66 as a novel tumor antigen with its strong tumorigenic ability might be a novel target for the early detection, prevention and treatment of tumor in the future.
Indoleamine 2,3-dioxygenase-1 (IDO1) expression by childhood acute myeloid leukemia restrains IFN-γ production by T cells and may portend an unfavorable prognosis (P2157)
Authors: Rutella Sergio , Folgiero Valentina , Masetti Riccardo , Pagliara Daria , Caruso Roberta , Vinti Luciana , Moretta Francesca , Pession Andrea , Locatelli Franco
Published: January 2023

Background: Indoleamine 2,3-dioxygenase 1 (IDO1) degrades tryptophan (TRP) into kynurenine (KYN), driving the development, stabilization and activation of regulatory T cells, and suppressing anti-tumor T-cell responses. Patients and methods: We investigated IDO1 expression and function in 22 children with acute myeloid leukemia (AML; median age at diagnosis: 8 years, range 1-16). IDO expression was assessed with Western blotting; TRP and KYN levels were measured with RP-HPLC. Results: Leukemia blasts from 10 out of 21 evaluable AML cases up-regulated IDO1 expression and function after in vitro challenge with IFN-γ, as indicated by higher KYN levels in supernatants of stimulated (24.3 μM/L, range 10.8-37.7) compared with unstimulated AML cells (1.3 μM/L, 0.9-2.7; p=0.0022). IDO induction was abrogated by STAT3 inhibitors; furthermore, co-immuno-precipitation assays suggested the occurrence of physical interactions between IDO and STAT3 in leukemia cells. IDO-expressing AML blasts restrained Th1 cytokine production by allogeneic CD8+ T cells, an effect that was partially abrogated by D,L-1-methyl-tryptophan, an IDO inhibitor. A Kaplan-Meier estimate of the 3-year overall survival indicated a clear benefit for IDO- AML cases compared with IDO+ ones (68% versus 33%). Conclusions: Blast cells from childhood AML express functional IDO1. From a therapeutic standpoint, interference with IDO1 activity may restore anti-leukemia Th1 immune responses and improve disease outcome.
Activated B cells infiltrate lung cancer (P2201)
Authors: Bruno Tullia , Kern Jeffrey , Slansky Jill
Published: January 2023

Lung cancer is the leading cause of cancer death, and although some novel therapies are emerging, we are interested in boosting antigen-specific responses. In a comprehensive survey of the immune infiltrate in non-small cell lung cancer (NSCLC) patients, we found that the total number of B cells (TIL-Bs) in the tumor versus the tumor-adjacent tissue was increased in comparison to other immune subsets, specifically T cells. NSCLC TIL-Bs are activated (CD21lo and CD69+), proliferate, and have a memory phenotype (CD27+). Preliminary functional studies indicate that these cells generate activation cytokines (IFN-γ and IL-12), which suggest that the TIL-Bs have an effector phenotype that could aid in targeting the tumor. Further, these cells secrete total IgG and may present antigen because of their juxtaposition to T cells at the site of the tumor. In addition, we are analyzing the surface expression of Fas-ligand and TRAIL on the TIL-Bs to determine if these cells could be involved in the direct killing of tumor cells. Because we are interested in uncovering the unique potential of these TIL-Bs, we are currently collecting cells from tumor and tumor-adjacent tissue for gene-expression profiling. By comparing these two populations to normal lung B cells, we aim to discover a unique gene or pathway in the TIL-Bs that can be targeted for an immunotherapy clinical trial in lung cancer patients.
Role of immunoglobulin heavy chain intronic enhancer Eμ in cell selection at the pre-B to immature B cell transition (P1437)
Authors: Peng Cheng , Eckhardt Laurel
Published: January 2023

We previously described an Eμ-dependent checkpoint for allelic exclusion, operating at the pre-B to immature B cell transition. We now show that Eμ functions at this transition to ensure that Igμ levels, and consequently B cell receptor (BCR) levels, reach a threshold required for positive selection into the immature B cell pool. We find that Ig-mediated signalling is reduced in pre-B cells expressing an Eμ-deficient IgH allele, as is the size of the emerging immature B cell compartment. Some Eμ-deficient B cells break the rules of allelic exclusion as “double-producers” to achieve higher BCR levels. We previously showed that B cells expressing only the Eμ-deficient allele underwent heightened light-chain editing, consistent with the hypothesis that deficient BCR signals were driving further light chain gene rearrangements in search of an IgH/IgL combination with superior signalling properties. In the current study, we tested this by providing Eμ-deficient mice with a pre-assembled VL gene. As predicted, both heavy chain allelic exclusion and the size of the immature B cell pool were affected. In addition, we found that the double-producers in allelic exclusion-defective mice include a subpopulation in which the BCR generated from the Eμ-deficient allele is autoreactive. These results demonstrate that Igμ levels in developing B cells have an important impact on the breadth of the BCR repertoire, the maintenance of allelic exclusion, and the emergence of autoimmune B cells.
Bcl-xL protects from UPR-associated apoptosis during plasma cell differentiation. (P1457)
Authors: Gaudette Brian , Iwakoshi Neal , Boise Lawrence
Published: January 2023

Understanding factors that control plasma cell survival is important for the development of therapeutic approaches to diseases including multiple myeloma and autoimmune disorders. Plasma cell differentiation requires activation of the unfolded protein response, a set of signaling pathways in which prolonged activation leads to apoptosis in other cell types. Plasma cells activate this pathway during differentiation suggesting compensatory survival signals are activated. Consistent with this hypothesis, we found that LPS-driven differentiation protects Bcl1 cells against tunicamycin-activated ER stress-induced death. LPS plus cytokine-induced differentiation was associated with an increase in Bcl-xL expression accompanied by a decrease in Mcl-1 or Bcl-2 in Bcl1 cells and primary B cells respectively. Moreover, differentiation-induced protection against ER stress was shown to be Bcl-xL-dependent as tunicamycin-induced death was restored by the addition of the Bcl-xL/Bcl-2 inhibitor, ABT-737. Importantly, we also showed that during differentiation, Bcl1 cells and primary B cells are Bcl-xL-dependent as ABT-737 resulted in significant apoptosis in the absence of any additional death signal. Finally, we found that differentiation was accompanied by a change in Bim binding from primarily Mcl-1 and/or Bcl-2 to Bcl-xL in differentiating cells. These data demonstrate that Bcl-xL induction protects from death signals activated during plasma cell differentiation.