bluuebunny/crossref_metadata_2025 huggingface dataset:
Discrimination of spectrally overlapping fluorochromes using Sony Corporations’s spectral analyzer (SP6800) (TECH1P.865)
Authors: Divekar Anagha , Lee MIchael , Kakuta Masaya , Nao Nitta , Motohiro Furuki , Yang Xifeng , Ransom John
Published: January 2023

A maximum of seven colors can be separated using traditional 2-laser flow cytometers and it is required to have the necessary band pass filters to separate these fluorochromes. In addition, these systems cannot differentiate between spectrally overlapping fluorophores such as PerCP and PerCP/Cy5.5; or FITC, PE and Alexa Fluor 514® (A514). SP6800, a novel spectral analyzer from Sony Corporation uses an innovative optics system that collects all emitted light and eliminates the need for band pass filters. We performed whole blood staining using a panel of 9 markers, which included significantly overlapping fluorochromes PerCP and PerCP/Cy5.5 and A514, PE and FITC and analyzed it on the SP6800. We were able clearly identify CD56+ (PerCP/Cy5.5), CD19+ (PerCP) and CD3+ (A514) cells. Further comparison of a 7-color panel of consisting of CD3 FITC, CD56 PE, CD19 PerCP, CD8 PE/Cy7, CD16 APC, CD4 Alexa Fluor 700® and CD62L APC/Cy7 on the BD LSRFortessa™ system and SP6800 gave similar results. Our data show that the SP6800 can perform all the analysis similar to a traditional cytometer with the added feature of separating spectrally overlapping fluorophores.
Optimizing adoptive cancer immunotherapy with γδ T cells through “pulse” zoledronate stimulation and IL-15 (TUM2P.886)
Authors: Nada Mohanad , Wang Hong , Workalemahu Grefachew , Morita Craig
Published: January 2023

Human γδ T cells expressing Vγ2Vδ2 TCRs monitor foreign- and self-prenyl pyrophosphate metabolites in isoprenoid biosynthesis to mediate immunity to microbes and cancer cells. Vγ2Vδ2 T cells have been used for adoptive cancer immunotherapy with some partial and complete remissions. Most clinical trials have used zoledronate to expand Vγ2Vδ2 T cells. Zoledronate inhibits farnesyl pyrophosphate synthase causing isopentenyl pyrophosphate to accumulate that then stimulates Vγ2Vδ2 T cells. Because constant zoledronate exposure is toxic, we hypothesized that a short "pulse" of zoledronate would reduce T cell toxicity because the monocytes that function as APC would preferentially take up zoledronate. We now find that "pulse" exposure of PBMC to zoledronate results in more uniform expansion of Vγ2Vδ2 T cells with higher purity and cell numbers along with better preservation of the early memory subsets that are the most effective for cancer immunotherapy. IL-15 was superior to IL-2 because it further preserved early (central) memory subsets. The Vγ2Vδ2 T cells also exhibited increased tumor cytotoxicity. Thus, “pulse” zoledronate exposure and substituting IL-15 for IL-2 maximizes the quantity and quality of expanded Vγ2Vδ2 T cells and represent improvements on the protocols being used in clinical trials. Moreover, because Vγ2Vδ2 T cells exhibit no autoimmunity or alloreactivity, they are potential recipients for the expression of chimeric antigen receptors.
Efficient identification of mutated cancer antigens recognized by T cells associated with durable tumor regressions (TUM2P.878)
Authors: Lu Yong-Chen , Yao Xin , Crystal Jessica , Li Yong , El-Gamil Mona , Gross Colin , Davis Lindy , Dudley Mark , Yang James , Samuels Yardena , Rosenberg Steven , Robbins Paul
Published: January 2023

Cancer immunotherapy with adoptive transfer of tumor infiltrating lymphocytes (TILs) represents an effective treatment for patients with metastatic melanoma, with the objective regressions in up to 72% of patients in three sequential pilot clinical trials. However, the antigen targets recognized by effective TILs remain largely unclear. Melanoma patients 2359 and 2591 both experienced durable complete regressions of metastases ongoing beyond five years following adoptive TIL transfer. A novel approach was developed to identify mutated T-cell antigens by screening a tandem minigene library, which comprised non-synonymous mutation sequences identified by whole-exome sequencing of autologous tumors. The screening of tandem minigene libraries resulted in the identification of mutated kinesin family member 2C (KIF2C) antigen as a target of TIL 2359, and mutated DNA polymerase alpha subunit B (POLA2) antigen as a target of TIL 2591. Both KIF2C and POLA2 have been found to play important roles in cell proliferation. These findings suggest that the minigene screening approach may facilitate the antigen repertoire analysis of tumor reactive T cells, and lead to the development of novel adoptive cell therapies based on the in vitro stimulation of T cells that recognize candidate mutated antigens derived from genes essential for the carcinogenesis.
Chronic alcohol consumption decreases the survival and compromises the antitumor immune response of mice bearing estrogen receptor positive E0771 breast cancer (TUM2P.898)
Authors: Zhang Hui , Zhang Faya , Zhu Zhaohui , Meadows Gary
Published: January 2023

Epidemiological data convincingly indicate that chronic alcohol consumption increases the risk of breast cancer, especially estrogen receptor positive (ER+) breast cancer in women. The effect of alcohol on antitumor immunity and survival remains to be elucidated. Herein, we implanted ER+ E0771 breast cancer cells into the mammary pad of female C57BL/6 mice consuming 20% w/v alcohol for three months and examined the effect of chronic alcohol consumption on tumor growth, dynamic changes in the antitumor immune response and host survival. Alcohol did not affect primary tumor growth or the body weight of tumor-bearing mice; however, it decreased survival. Alcohol did not alter the percentage of MDSC, Treg, CD4+ T or CD8+ T cells in the spleen and blood, but increased theCD4/CD8 ratio. B cells decreased during tumor growth, and iNKT cells increased in the blood of alcohol-consuming mice. In non-tumor-bearing mice, alcohol increases IFN-γ-producing CD8+ T cells and NK cells. Four weeks after E0771 inoculation, the percentage of IFN-γ-producing CD8+ T cells and NK cells were lower in alcohol-consuming mice compared to water-drinking mice. Alcohol consumption also decreased survival of mice immunized with an E0771 cell lysate and boosted with αGalCer compared to water-drinking counterparts. Collectively, chronic alcohol consumption compromises antitumor immunity and decreases the survival of mice bearing ER+ breast cancer.
Breast cancer diagnosis by high throughput sequencing of T cell receptors from peripheral blood samples (TUM2P.905)
Authors: Wang Chunlin , Alleyne Jessica , Yang Qunying , Byrne-Steele Miranda , Ng Enders , Kwong Ava , Shin Vivian , Han Jian
Published: January 2023

T cells play vital roles in the identification and elimination of transformed cells. It might be possible to diagnose cancer by reading imprints on the immune system made by cancer cells. High-throughput immune repertoire sequencing allows the depiction of T cell repertoire details at a nucleotide resolution and enables us to search for cancer-specific biomarkers. RNA was isolated from whole blood in breast cancer and matched controls and the CDR3 region of the TCR beta chain amplified and sequenced by Illumina next-generation sequencing platform. Initial studies showed around 200 breast cancer-specific CDR3 sequences. Two thirds of those CDR3 sequences show a pattern of convergent evolution, i.e., the same CDR3 peptide sequence is coded by different sequences. To leverage those disease-specific CDR3’s for diagnostics, we developed a novel metric called immune repertoire sharing index (SI) to measure the similarity between a repertoire and the disease library. With SI, we were able to predict breast cancer cases with both the sensitivity and the specificity around 90%. The diagnostics process requires only 10ml peripheral blood. We hypothesized that the immune repertoire sequencing and sharing index method could be generalized to other diseases. An ‘ultimate diagnostics’ may be emerged where taking one sample, with one procedure; hundreds of diseases can be diagnosed by evaluating immune repertoires.
An inflammatory T cell signature predicts obesity-associated type 2 diabetes (HUM3P.262)
Authors: Ip Blanche , Cilfone Nicholas , Zhu Min , Kuchibhatla Ramya , Azer Michel , McDonnell Marie , Apovian Caroline , Lauffenburger Douglas , Nikolajczyk Barbara
Published: January 2023

T cell inflammation plays pivotal roles in obesity-associated type 2 diabetes (T2D). Multiple T cell subsets, including Th1, Th2, Th17 and regulatory T cells are critical for the development of insulin resistance in animal models of T2D. However, the relative importance of T cell subsets in human disease pathogenesis remains a critical gap in knowledge with important implications for defining the most potent inflammatory mediators of T2D. To identify T cell subsets that disproportionately contribute to T2D-associated inflammation, we broadly quantified T cell cytokine production by circulating or adipose-associated immune cells from obese subjects clinically defined as non-T2D, pre-T2D or T2D. Cytokines traditionally associated with specific T cell subsets, including IL-2 (Th1), IL-4/10/13 (Th2) and IL-17A/F (Th17), were produced at highest amounts by T2D samples. We combined cytokine outcomes with multivariate mathematical approaches to show that T cell cytokine profiles predicted T2D status with >85% accuracy. Furthermore, Th17 signature cytokines were more powerful than either Th1 or Th2 cytokines for differentiation of non-T2D from T2D samples. We conclude that Th17 cells are an underappreciated source of T cell cytokines that support inflammation and predict human T2D. Study outcomes unify contradictory claims of the importance of single cytokines and justify including Th17 cells as druggable sources of inflammation in human T2D.
MSPrecise: a new diagnostic test for relapsing-remitting multiple sclerosis by cerebrospinal fluid B cell sequencing (HUM3P.267)
Authors: Rounds William , Salinas Edward , Wilks Tom , Levin Mikhail , Ligocki Ann , Ionete Carolina , Pardo-Villamizar Carlos , Greenberg Benjamin , Bigwood Douglas , Eastman Eric , Cowell Lindsay , Monson Nancy
Published: January 2023

Patients with relapsing-remitting multiple sclerosis (RRMS), or who will convert to RRMS, have cerebrospinal fluid (CSF) B cells that accumulate specific replacement mutations in their rearranged VH4 gene segments as identified by single-cell Sanger sequencing. To harness this observation in a clinical setting, we have developed a diagnostic test to extract and score this genetic information from bulk CSF lymphocytes by next-generation DNA sequencing (NGS). Our current aim is to determine the accuracy of this test on a cohort of patients with varying times of RRMS onset and disease modifying therapies, as well as on a diverse cohort of patients with other neurological diseases (OND) that generally mimic the presentation of MS. Our initial testing on 39 patients has shown an accuracy of 84% in distinguishing RRMS from OND patients. As we continue to expand the size of the cohorts being tested, we will also focus on identifying differences between sub-cohorts of patients at different stages of RRMS or with particular OND diagnoses. With a growing need for a faster and more accurate diagnosis of RRMS, MSPrecise may be a useful addition to the existing diagnostic toolkit that clinicians use to achieve this goal.
B lymphocytes limit the severity of emphysema caused by N. brasiliensis infection in mice (INC4P.332)
Authors: Chen Fei , Wu Wenhui , Millman Ariel , Palma Mark , Gause William
Published: January 2023

The intestinal nematode parasite, Nippostrongylus brasiliensis (Nb), transiently resides in the lung causing lung damage, resulting in emphysema. However, the mechanisms contributing to this chronic lung pathology remain not well-defined. Here we show that emphysema develops as early as 7 days after Nb inoculation, as measured by alveolar mean linear chord length and is associated with increased neutrophil elastase (NE) and macrophage matrix metalloproteinase 12 (Mmp12) gene expression in the lung. Transfer of sorted Nb-primed neutrophils or macrophages to naïve recipients results in emphysema, indicating that either of these populations can trigger lung emphysema in naïve mice. Parasite infection also results in more severe emphysema in B cell deficient Jh-/- mice compared to wild-type mice or FcRγ-/-mice and is associated with higher numbers of neutrophils and macrophages, as well as increased expression of NE and Mmp12 in the lung. Transfer of B lymphocytes from wild-type mice to the Jh-/- mice can restore control of emphysema. These data suggest that Nb-primed neutrophils and macrophages contribute to lung emphysema. Furthermore, parasite-induced B cells exert regulatory effects that can markedly reduce the severity of emphysema.
FcRγ-chain, Grb2 and SHP-1 are invovled in mannose receptor (CD206)-mediated signaling in relation to tuberculosis (INC4P.329)
Authors: Rajaram Murugesan , Ni Bin , Arnett Eusondia , Abul Azad , Schlesinger Larry
Published: January 2023

The mannose receptor (MR, CD206) is highly expressed on human macrophages. It is a major pattern recognition receptor for pathogens, mediates phagocytosis and endocytosis, and regulates inflammatory signaling pathways. We have shown that the MR mediates Mycobacterium tuberculosis (M.tb) phagocytosis by human macrophages which leads to up-regulation of PPARγ expression and a delay in phagosome-lysosome (P-L) fusion. However, the associated receptor-specific downstream signaling molecules are unknown. We assayed for MR-associated proteins by MALDI-TOF and Western blot using a pull down assay with macrophage lysates and phosphorylated and un-phosphorylated MR cytoplasmic tail peptides. We have identified that FcRg-chain interacts with the MR and this interaction is necessary for MR surface exposure on human monocyte-derived macrophages (MDMs). Furthermore, M.tb infection of MDMs leads to phosphorylation of MR tyrosine residues and recruitment of Grb2 which initiates the Rac/Pak/Cdc-42 signaling cascade required for phagocytosis, and activation of MAP kinases and the PI3k/Akt pathway. MR activation also recruits and activates SHP-1. SHP-1 blockade increases M.tb P-L fusion and inflammatory cytokines. In conclusion, we have identified key MR signaling molecules and regulatory pathways which initiate phagocytosis and other immune-related functions in human macrophages.
Identification and characterization of phosphodiesterases that specifically degrade 3’3’-cyclic GMP-AMP (INM2P.358)
Authors: Jiang Zhengfan
Published: January 2023

Cyclic di-nucleotides act as intracellular second messengers, modulating a variety of cellular activities including innate immune activation. Whereas phosphodiesterases (PDEs) hydrolyzing c-di-GMP and c-di-AMP have been identified, no PDEs for cGAMPs have been reported. Here we identified the first three cGAMP specific PDEs in V. cholerae (herein designated V-cGAP1/2/3 respectively). V-cGAPs belong to HD-GYP domain containing proteins, and specifically break 3’3’-cGAMP, but not other forms of cGAMP. 3’3’-cGAMP was linearized to 5’-pApG by all three PDEs, and was further hydrolyzed to 5’-ApG by V-cGAP1, which functions as both a phosphodiesterase and a 5’-nucleotidase. In vivo experiments demonstrated that V-cGAPs play non-redundant roles in cGAMP degradation. The high specificity of V-cGAPs on 3’3’-cGAMP suggests the existence of specified PDEs for other cGAMPs, including 2’3’-cGAMP in mammalian cells; and the absolute requirement of GYP motif for 3’3’-cGAMP degradation indicates that HD-domain containing PDEs in eukaryotes are probably not able to hydrolyze cGAMPs. The fact that all V-cGAPs attack 3’3’-cGAMP on one specific phosphodiester bond suggests that PDEs for other cGAMPs would utilize the similar strategy. These results will provide valuable information to search for and characterize the mammalian 2’3’-cGAMP-specific PDEs in future studies.
Despite KLRG1 expression, highly cytotoxic ThEO/TcEO polarized T cells efficiently establish immunologic memory (LYM5P.662)
Authors: Bartkowiak Todd , Ai Midan , Ager Casey , Curran Michael
Published: January 2023

Targeting immune inhibitory (CTLA-4, PD-1) or stimulatory (OX-40, 4-1BB) co-receptors has proven a potent anti-cancer therapeutic strategy, eliciting robust anti-tumor T cell responses and compelling pre-clinical and clinical data in a variety of cancers. We have previously shown in pre-clinical models that 4-1BB agonist antibodies engender strong anti-tumor T cell responses, imparting enhanced survival benefit in addition to evoking durable tumor regression. This impressive anti-tumor effect is due, in part, to the unique ability of 4-1BB co-stimulation to differentiate a novel subset of T cells that acquires a highly cytotoxic effector profile typically only seen in NK cells. This subset-dubbed ThEO (CD4+) or TcEO (CD8+)-is driven by the T-box transcription factor Eomesodermin, and is further characterized by expression of the inhibitory receptor KLRG1. We show here that, despite their strong effector function and expression of several co-inhibitory receptors (PD-1, TIM-3, KLRG1), the ThEO/TcEO phenotype establishes immunological memory and recalls with high frequency upon secondary tumor challenge with potent anti-tumor effector function. Further, these cells manifest characteristics of central memory (Tcm), effector memory (Tem), as well as stem cell memory (Tscm), and reside in multiple memory niches. These findings provide mechanistic insight into the process of memory formation and give evidence for the capacity of ThEO/TcEO cells to prevent tumor recurrence.
CD4+ CD44v.low cells are peripheral CD4+ T cell precursors that are distinct from recent thymic emigrants and stem cell-like memory cells (LYM6P.713)
Authors: Narsale Aditi , Zhao Chunfang , Marrero Idania , Davies Joanna
Published: January 2023

CD4+ CD44v.low cells are peripheral precursor T cells that inhibit lymphopenia by generating a large CD4+ T cell pool containing balanced numbers of naïve, memory, and regulatory Foxp3+ cells with a diverse TCR repertoire. They can also inhibit cachexia, a profound wasting syndrome that is associated with lymphopenia and immune dysregulation. Recent thymic emigrants (RTE) and stem cell-like memory T cells (TSCM) can also replenish a T cell pool. In this study we formally test whether CD44v.low cells are the same population as RTE and TSCM. Our data show that, in contrast to RTE, CD44v.low cells express high levels of CD45RB and low levels of CD24. Moreover, CD44v.low cells isolated from mice devoid of RTE retain their capacity to repopulate lymphopenic mice with naïve and memory cells and Foxp3+ Tregs. In addition, CD44v.low cells do not express IL-2Rb, Sca-1, and CXCR3, the phenotypic hallmarks of TSCM. Further characterization of the cells generated by CD44v.low cells shows that they secrete significantly more Th2-type cytokines IL-4 and IL-6, significantly less Th17/Th22-type cytokines IL-17A and IL-22, but equivalent Th1-type cytokines IFN-g and TNF-a compared to cells generated by their naïve cell counterparts. Overall, these data demonstrate that CD44v.low cells are neither RTE nor TSCM. Moreover, their capacity to promote a balance cytokine profile might explain their ability to inhibit cachexia.
Human pDCs express the C-type Lectin receptor Dectin-1 and uptake and kill Aspergillus fumigatus spores in vitro (MPF4P.734)
Authors: Maldonado Samuel , Dai Jihong , Singh Sukhwinder , Mwangi David , Rivera Amariliz , Fitzgerald-Bocarsly Patricia
Published: January 2023

Plasmacytoid dendritic cells (pDCs) are the body’s most potent producers of type-1 IFNs, and are key responders to viruses like HIV, serving as an important link between the innate and adaptive immune responses. Although they are mainly anti-viral, evidence suggests pDCs also play an integral part in anti-fungal immunity. We demonstrated that human pDCs express the C-type Lectin receptor (CLR), Dectin-1, and that it is further upregulated after stimulation with HSV, Influenza, and the synthetic DNA ODN, CpG-A. In contrast, we did not find expression of Dectin-2, another CLR, on pDCs. Dectin-1 recognizes β-linked glucans, a component of the cell wall of opportunistic fungi like Aspergillus, Pneumocystis, and Candida, and is involved in immune functions including the uptake of fungi, induction of cytokine production, and direction of T-helper cell differentiation. Using Fluorescent Aspergillus Reporter (FLARE)-labelled A. fumigatus spores, which allow us to detect and differentiate between live and dead spores using flow cytometry, we showed that human pDCs can phagocytose and kill Aspergillus spores in vitro within a six-hour time frame. We hypothesize that Dectin-1 is the receptor responsible for this uptake. Although pDCs are classically considered IFN producers, stimulation with spores did not result in IFN-α production by pDC. Results from this ongoing study will shed further light on the role of human pDCs in anti-fungal immunity.
AIM2 and NLRP3 inflammasome activation provide dual surveillance to confer protection against Aspergillus infection (MPF4P.728)
Authors: Karki Rajendra , Man Si Ming , Kanneganti Thirumala-Devi
Published: January 2023

Pulmonary aspergillosis is a leading cause of infection-associated mortality in immunocompromised individuals. Recognition of Aspergillus by pattern recognition receptors drives innate immune responses in the host. However, the role of cytosolic receptors in inflammasome activation during Aspergillus infection is unclear. Here, we showed that two different inflammasome receptors, AIM2 and NLRP3, play redundant roles in inflammasome activation in response to A. fumigatus infection. While mice deficient in AIM2 or NLRP3 alone showed similar susceptibility compared to WT mice, mice doubly deficient for both AIM2 and NLRP3 receptors were highly susceptible to Aspergillus infection. The increased susceptibility in AIM2 and NLRP3 DKO mice was associated with significantly higher Aspergillus burden and concomitantly reduced inflammatory responses in the lungs. Bone marrow chimera study further confirmed that AIM2 and NLRP3 play vital role in both stromal and hematopoietic compartments during Aspergillus infection. Mechanistically, activation of these receptors initiates assembly of ASC, caspase-1 and caspase-8, for effective processing of IL-1β and IL-18. In agreement, mice lacking caspase-8, IL-1β or IL-18 were highly susceptible to Aspergillus infection in comparison to WT controls. Together, these findings reveal that dual surveillance system provided by both AIM2 and NLRP3 inflammasome receptors is required to provide anti-fungal host responses against A. fumigatus.
Role of plasma high-molecular-weight-kininogen in the pathogenesis of inflammatory bowel disease (MUC5P.748)
Authors: Wang Bo , Wu Yi
Published: January 2023

High-molecular-weight kininogen (HK) is a major component of plasma kallikrein-kinin system (KKS), its cleavage products bradykinin and HKa are proinflammatory. Clinical observations have shown that KKS activation and HK cleavage are involved in inflammatory bowel disease (IBD). However, the pathogenic role of HK in IBD have never been characterized using a genetic approach. Our goal is to characterize the role of HK in the pathogenesis of IBD，using HK-deficient (Kng1-/-) mice we recently generated. Dextran sulfate sodium (DSS)-induced colitis in mice were induced by 2.5% (wt/vol) DSS in drinking water for 7 days. Plasma bradykinin level in wild-type (WT) mice was elevated on day 7 (p<0.001). Weight loss index and disease activity index (DAI) of DSS-treated Kng1-/- mice were significantly lower than those of WT mice (p<0.05). Kng1-/- mice had a decrease in colonic cytokines (TNF-α, IL-1β, IL-6 and INF-γ) and myeloperoxidase (MPO) levels (p<0.001). As indicated by H&E staining, Kng1-/- mice exhibited a reduced colonic tissue damage and neutrophil infiltration in colonic lamina propria (p<0.001). Similar with Kng1-/- mice, the mice lacking double bradykinin receptors (B1RB2R-/-) exhibited an amelioration of DSS-induced colitis, including the amelioration of weight loss, DAI, colonic tissue damage, and colonic cytokine levels. This study provides the first genetic evidence demonstrating that HK and its cleavage product bradykinin play a critical role in the pathogenesis of IBD.
Intrathymic transplantation of young, engraftable thymic epithelial cells promotes the growth and regeneration of the aging thymus (TRAN1P.932)
Authors: KIM MIJEONG , Miller Christine , Shadrach Jennifer , Wagers Amy , Serwold Thomas
Published: January 2023

The thymus reaches its maximum size early in life and then begins to shrink, producing fewer T cells with increasing age. This thymic decline is thought to contribute to age-related T cell lymphopenias and hinder T cell recovery following bone marrow transplantation. While several cellular and molecular processes have been implicated in age-related thymic involution, their relative contributions are not known. Using heterochronic parabiosis, we observe that young circulating factors are not sufficient to drive regeneration of the aged thymus. In contrast, we find that resupplying young, engraftable thymic epithelial cells to an aged or defective thymus leads to thymic regrowth and renewed T cell production. Intrathymic transplantation and in vitro colony forming assays reveal that the engraftment and proliferative capacities of thymic epithelial cells diminish early in life, whereas the receptivity of the thymus to thymic epithelial cell engraftment remains relatively constant with age. These results support a model in which thymic growth and subsequent involution are driven by cell intrinsic changes in the proliferative capacity of thymic epithelial cells, and further show that young thymic epithelial cells can directly drive aged thymic regeneration.
Acute graft-versus-host disease impairs anti-viral immunity and leads to fulminant CMV disease (TRAN1P.937)
Authors: Degli-Esposti Mariapia , Wikstrom Matthew , Kuns Rachel , Fleming Peter , Tey Siok-Keen , Andoniou Christopher , Hill Geoffrey
Published: January 2023

Viral infection is a common, life-threatening complication of allogeneic bone marrow transplantation (BMT), particularly in the presence of graft-versus-host disease (GVHD). The inability to mount protective anti-viral immunity is well recognized, yet the defects remain unclear. Using cytomegalovirus (CMV) as the prototype pathogen, we delineated the cause of the disease that arises following infection, as well as the mechanisms that lead to the inability to mount effective anti-viral immunity in this setting. While infection was self-limiting after syngeneic BMT, CMV infected mice with GVHD developed a fulminant necrotizing hepatitis directly associated with extensive CMV cytopathy. GVHD induced profound defects in dendritic cells (numerical and resistance to infection) that profoundly impaired viral antigen presentation, preventing the priming of CMV-specific CD8+ T cells. Changes indicative of T cell exhaustion were also observed. The transfer of virus-specific CD8+ T cells pre-infection circumvented the priming defect and provided protective immunity. Notably, efficacious anti-viral immunity could still be generated when T cells were transferred at the time of BMT, a month prior to infection, despite concurrent GVHD. These data demonstrate the importance of avoiding GVHD when reconstructing anti-viral immunity after BMT, and highlight the mechanisms by which the adoptive transfer of virus-specific T cells overcome the endogenous defects in priming invoked by GVHD.
Stem cell and novel biologic therapies to enhance functional recovery in vascular composite allotransplantation (TRAN1P.922)
Authors: Kniery Kevin , DeHart Mary , Brown Tommy , Salgar Shashikumar
Published: January 2023

Introduction: Vascular composite allotransplantation (VCA) such as limb and face require successful nerve regeneration and reinnervation of graft motor and sensory targets for optimal clinical outcome. Methods: Lewis (RT1.Al) rat was anesthetized and individual sciatic nerve branches (tibial, peroneal and sural) of the right hind-limb were transected. After one hour the epineurium of the transected nerves were approximated by 10-O sutures. Mesenchymal Stem Cells (MSC; 5x106; passage ≤6), G-CSF (50 µg/kg), Dihexa (2 mg/kg) or Vehicle were administered locally and intravenously. Results: Rat MSC expanded ex vivo were CD29+, CD90+, CD34-, CD31-, CD45low, MHC Class I+, and Class II-; and were pluripotent. At two weeks post nerve repair, total sensory nerve function in all groups was ~1.5 on a scale of Grade 0-3 (0=No function; 3=Normal function). By 8 weeks sensory function was ≥ 2.5 in all groups studied (n≥6); slightly lower in the vehicle group. Peroneal sensory function appeared as early as one week (≥2) but not tibial or sural function (~0.5). At five weeks, the sciatic nerve function index (SFI) a measure of motor function (0=Normal function; -100=Nonfunctional) was ~ -50 in G-CSF, and ~ -80 in MSC, Dihexa, and Vehicle treated groups (n≥6). By 12 weeks SFI in G-CSF group was markedly improved (-25). Histology and molecular studies are ongoing. Conclusions: MSC and G-CSF appear to promote both sensory and motor nerve functions in nerve transection repair model.
Nrf2 is a critical regulator of allogeneic T cell function and acute graft-versus-host disease (TRAN1P.923)
Authors: Tsai Jennifer , Dudakov Jarrod , Velardi Enrico , Shono Yusuke , Smith Odette , Wertheimer Tobias , Jenq Robert , Hanash Alan , Van Den Brink Marcel
Published: January 2023

Nuclear factor erythroid-derived 2-like 2 (Nrf2) is an ubiquitously expressed transcription factor, well-known as a master regulator of cellular redox pathway. A recent paper showed that Nrf2 can modulate Th1/Th2 axis in vitro. Here, we evaluated the relative contribution of Nrf2 to alloreactive T cells in the context of allogeneic bone marrow transplant (allo-BMT). We found that Nrf2-deficiency in donor T cells led to significantly less acute graft-versus-host disease (GVHD) in both MHC-mismatched and MHC-matched, minor antigen mismatched murine models. Surprisingly, Nrf2-/- T cells demonstrated comparable levels of proliferation, activation, and similar sensitivity to apoptosis during both early expansion and contraction phase of acute GVHD. However, Nrf2-/- T cells showed markedly reduced infiltration and tissue damage in the intestines, in keeping with their significantly lower expression of α4β7 integrin. Serum cytokine analysis on day 7 of allo-BMT showed increased IFNγ, IL-4 and IL-5 levels in the recipients of Nrf2-/- allo-T cells without a concomitant rise in TNFα, IL-13 or IL-17 production, while intracellular staining showed a marked increase in the proportion of CD25+Foxp3+ regulatory T cells in Nrf2-/- donor T cells. Taken together, we identified a novel role of Nrf2 in T cell function in the context of allo-BMT. Our findings suggest that targeted manipulation of Nrf2 activities in alloreactive T cells may offer a useful clinical strategy to mitigate GVHD.
Imprime PGG, a yeast β-glucan immunomodulator has the potential to modulate the subtypes of immunosuppressive M2 macrophages (TUM6P.962)
Authors: Bose Nandita , Qiu Xiaohong , Jonas Adria , Chan Anissa , Kangas Takashi , Graff Jeremy
Published: January 2023

Imprime PGG (IPGG) is a yeast β-1,3/1,6 glucan that, when complexed with endogenous anti-β-glucan antibodies, binds and primes neutrophils and monocytes to exert cytotoxic activity against antibody-targeted cancer cells via a complement receptor 3-dependent mechanism. Randomized Phase 2 clinical trials of IPGG in the 1st-line treatment of stage IV non-small cell lung cancer have shown promising efficacy in terms of both objective tumor response and survival. Recent in vitro human mechanistic work revealed that IPGG inhibits the polarization of immunosuppressive M2 macrophages. These studies used macrophage-colony stimulating factor (M-CSF) as the polarizing cytokine. Given that the macrophages undergo specific differentiation depending on the cytokine milieu in the local tissue microenvironment, the objective of the current study was to further evaluate the effect of IPGG on the other subsets of M2 macrophages, including M2a (elicited by M-CSF and IL-4) and M2c (elicited by M-CSF and IL-10). The results demonstrated that IPGG induced phenotypic changes on both type of macrophages, including upregulation of PD-L1. In terms of functional activity, IPGG enhanced the ability of M2a macrophages to enhance CD4 T cell proliferation, and the ability of M2c macrophages to phagocytize tumor cells. These results, along with previous results, demonstrate that IPGG has a strong potential to downmodulate the immunosuppressive tumor microenvironment shaped by tumor-associated macrophages.
Alteration in immuno-suppressive macrophage responses is associated with inhibition of vacuolar ATPase activity in breast cancer (TUM6P.968)
Authors: Katara Gajendra , Kulshrestha Arpita , Jaiswal Mukesh , Gilman-Sachs Alice , Beaman Kenneth
Published: January 2023

Vacuolar ATPases (V-ATPase) are important for tumor growth and metastasis. A novel, peptide of a2 isoform of V-ATPase (a2V) has been shown to have an immunoregulatory role in the tumor microenvironment by controlling the maturation of monocytes towards a TAM phenotype. This activity caused us to further investigate the role of a2V in cancer progression. Our results showed that inhibition of a2V in tumor cells have a significant growth reducing effect on the tumor and its associated microenvironment. Tumor volume analysis revealed that tumor growth in mice inoculated with a2V knocked down (sh-a2) 4T-1 cells was delayed up to ten fold compared to control (mean±SD: 1.3 ± 1.15 vs 13.96 ± 4.06, P>0.01) at day 8 after inoculation. When compared by flow cytometry and immuno-histochemistry, a decreased number (60%) of TAM (F4/80+CD206+) in cell suspensions from sh-a2 generated tumors and an increase in macrophage with anti-tumor activity (F4/80+CD11c+) was observed. Further, The expression of TAM associated molecules (mannose receptor-1 or CD206, IL-10, Arginase-1, matrix metalloproteinase and vascular endothelial growth factor) were significantly down regulated in tumors from sh-a2 inoculated mice compared to control (P<0.01). These findings demonstrate that the a2V from tumor cells can directly alter the resident TAM population and immune responses in the tumor microenvironment and hence may constitute novel target to control tumor growth through host immune system.
SOCS3 deficiency in myeloid cells promotes tumor development: involvement of STAT3 activation and myeloid-derived suppressor cells (TUM6P.1000)
Authors: Yu Hao , Benveniste Etty , Qin Hongwei
Published: January 2023

Suppressor Of Cytokine Signaling (SOCS) proteins are negative regulators of the JAK/STAT pathway, and generally function as tumor suppressors. The absence of SOCS3 in particular leads to heightened activation of the STAT3 transcription factor, which has a striking ability to promote tumor survival while suppressing anti-tumor immunity. We report for the first time that genetic deletion of SOCS3 specifically in myeloid cells significantly enhances tumor growth, which correlates with elevated levels of myeloid-derived suppressor cells (MDSC) in the tumor microenvironment, and diminished CD8+ T-cell infiltration in tumors. Furthermore, SOCS3-deficient bone-marrow-derived cells exhibit heightened STAT3 activation and preferentially differentiate into the Gr-1+CD11b+Ly6G+ MDSC phenotype. Importantly, we identify granulocyte-colony stimulating factor (G-CSF) as a critical factor secreted by the tumor microenvironment that promotes development of MDSC via a STAT3-dependent pathway. Abrogation of tumor-derived G-CSF reduces the proliferation and accumulation of Gr-1+CD11b+ MDSC and inhibits tumor growth. These findings highlight the critical function of SOCS3 as a negative regulator of MDSC development and function, via inhibition of STAT3 activation.
Antibody blockade of semaphorin 4D promotes infiltration of activated tumor infiltrating leukocytes and inhibits tumor growth. (TUM7P.1030)
Authors: Klimatcheva Ekaterina , Evans Elizabeth , Bussler Holm , Torno Sebold , Mallow Crystal , Winter Laurie , Reilly Christine , Veeraraghavan Janaki , Jonason Alan S. , Scrivens Maria , Kirk Renee , Giralico Susan , Howell Alan , Leonard John , Paris Mark , Fisher Terrence , Smith Ernest , Zauderer Maurice
Published: January 2023

Semaphorin 4D (SEMA4D, CD100) and its receptor plexin-B1 are broadly expressed in cancer and expression correlates with invasive disease. A novel function of SEMA4D, regulating immune cell infiltration and activity in the tumor microenvironment (TME), is described. Antibody neutralization of SEMA4D enhances recruitment of activated antigen presenting cells and lymphocytes into the TME, shifting the balance of cells and cytokines toward increased Th1 and reduced immunosuppression. This orchestrated change in the tumor architecture was associated with durable tumor rejection and immunologic memory in several syngeneic tumor models. The immunomodulatory activity can be enhanced by combination with other immunotherapies. Strikingly, in some models, the combination of anti-SEMA4D antibody with immune checkpoint blockade acts synergistically to reverse tumor growth. Inhibition of SEMA4D represents a novel mechanism and therapeutic strategy to promote functional immune infiltration into the tumor and inhibit tumor progression. A humanized antibody, VX15/2503, has completed a Phase I prospective multiple ascending dose trial in 42 adult patients with advanced refractory solid tumors, in which the highest doses were well tolerated. Patients with the longest duration of treatment, 48-55 weeks, included colorectal, breast, and a papillary thyroid patient, who had a partial response by RECIST. A phase 1b/2a trial of combination therapy is planned.
Increased expression of anti-oxidant molecule Thioredoxin-1 on T cells improves immunotherapeutic control of tumors (VAC7P.1039)
Authors: Kesarwani Pravin , Thyagarajan Krishnamurthy , Soloshchenko Myroslawa , Chatterjee Shilpak , Maulik Nilanjana , Mehrotra Shikhar
Published: January 2023

Adoptive transfer of tumor epitope reactive T cells is a promising strategy to control tumor growth. However, chronically stimulated T cells expanded for adoptive cell transfer (ACT) are susceptible to cells death in an oxidative tumor microenvironment. Since oxidation of cell surface thiols (c-SH) also alters functionality of proteins, we hypothesized that increased level of thioredoxin, an anti-oxidant molecule that facilitates reduction of proteins by cysteine thiol-disulfide exchange, in T cells will result in sustained anti-tumor function. We thus crossed Trx1 transgenic mice with gp100 reactive TCR (Pmel) to generate Pmel/Trx mice. The Trx overexpressing transgenic T cells expressed higher thiols that inversely correlated with ROS, and susceptibility to TCR restimulation or H2O2 mediated cell death. These Trx1 expressing T cells showed CD62Lhi central memory-like (TCM) phenotype with reduced effector function (IFNglo2-NBDGlo), and were less effective in controlling tumor upon adoptive transfer. However, adoptive transfer of Pmel cells mixed with Pmel/Trx1 cells in 1:1 ratio markedly improved tumor control as compared to the mice that were transferred Pmel or Pmel/Trx T cells alone. It is likely that Pmel immediate effectors and Pmel/Trx cells with T-stem cell memory phenotype (i.e. CD62LhiCD44loCD122+Sca1hi), contributed to the improved tumor control. Thus, strategies to increase anti-oxidant capacity of anti-tumor T cells could have immunotherapeutic implications.
BscF as a vaccine candidate for Bordetella pertussis infection (“Whooping Cough”) (VAC9P.1064)
Authors: Alvine Travis , Ganske Alec , Nilles Matthew , Bradley David
Published: January 2023

Bordetella pertussis, a Gram- bacterium, causes pertussis (“Whopping Cough”). Despite widespread vaccination, 48 million cases of pertussis occur annually worldwide. The resurgence of pertussis is linked to antigenic divergence of circulating pertussis and waning and/or ineffective immunity induced by the current acellular vaccine. YscF, a Yersinia protein that, in multimeric form, makes up the Type III Secretion System (T3SS) needle, has been shown to be both an excellent protective antigen for Y. pestis, as well as a strong adjuvant via TLR signaling. We characterized here immune responses induced by BscF, the Bordetella homolog of YscF. BscF activated NF-κB through both TLR-2 and TLR-4 in vitro. Moreover, BscF induced IL-1β and IL-12 secretion in human THP-1 cells and bone marrow-derived macrophages. In vivo, BscF immunization in alum induced both a Th1 and Th17 response, with stimulated CD4+ T cell secreting both IFN-γ and IL-17A. Interestingly, in vivo BscF (in alum) also induced significant titers of BscF-specific antibody of all IgG isotypes (1, 2a, 2b, 3), suggesting the induction of both Th1 and Th2 responses. In toto we demonstrated that BscF induces a strong humoral and cell-mediated protein-specific immune response. This, along with the adjuvant characteristic and external structural location of BscF, suggests that BscF may provide an excellent addition to the current acellular vaccine to yield a stronger and/or more persisted protection against pertussis.
Influenza-A virus specific Epstein-Barr virus lytic memory CD8 T cells in EBV sero-negative middle age adults (VIR5P.1147)
Authors: Mishra Rabinarayan , Watkin Levi , Aslan Nuray , Gil Anna , Luzuriaga Katherine , Selin Liisa
Published: January 2023

For unknown reasons 5-10% of middle-aged adults (>35 years) remain EBV-sero-negative when the virus infects the vast majority of people, and is actively shed at high titers during chronic infection. Here we show that EBV-sero-negative (EBV-SN) HLA-A2+ middle-aged adults possess a unique IAV-M158-66(GIL) memory CD8 T cell response that cross-reacts with EBV lytic epitopes. The five tested HLA-A2+ EBV-SN middle-aged adults had a significantly increased IAV-M158-66(GIL) tetramer+ CD8 frequency compared to adult EBV sero-positive donors. Upon exposure to EBV antigens in vitro both IAV- M1(GIL) /EBV-BMLF1280-288(GLC) and IAV- M1(GIL) /EBV-BRLF1109-117(YVL), functionally cross-reactive CD8+ responses could be detected in the peripheral blood of middle-aged EBV-SN donors, while only weakly IAV-(M1)/EBV-(YVL) cross-reactive responses were detected in 4 out of 6 teenage EBV-SN donors. These IAV-M1-GIL-specific CD8 T cells in middle-aged EBV-SN adults expanded dramatically to EBV lytic antigens and produced cytokines at high functional avidity. They lysed EBV-infected targets. Additionally, these cross-reactive cells had an oligo-clonal T cell receptor repertoire different than EBV sero-positive donors. Taken together these data suggest that an altered cross-reactive T cell repertoire could mediate protective immunity against viral infection. Our results imply that sero-negative adults might have the ability to resist viral infection via heterologous immunity.
Molecular regulation of host inflammation during influenza A virus infection (VIR5P.1155)
Authors: Yager Eric , Reid Bethany , Visconti Rachel , Harton Jonathan
Published: January 2023

The innate immune system recognizes influenza A viruses through multiple mechanisms, with recent studies revealing a critical role for the cytoplasmic NLRP3 inflammasome complex which promotes the maturation of the pro-inflammatory cytokine interleukin 1-β (IL-1β). Virus-induced NLRP3 inflammasome activation is critical for host defense and tissue repair during flu infection. Conversely, it is also evident that excessive NLRP3 inflammasome activity, especially during infection by highly pathogenic or pandemic strains of virus, can result in dysregulated inflammation that is detrimental to the host. Recent studies have identified a small molecule (pyrin-only protein 2 or POP2) that blocks the formation and activation of inflammasome complexes in humans. Because POP2 can interact with the pyrin domain of NLRP3, we hypothesize that POP2 functions to regulate the robust NLRP3-dependent IL-1β responses elicited during human IAV infection. Data from our continuing studies indicate that POP2 gene expression and function “cycles” with the expression of pro-IL-1β in PMA-differentiated human THP-1 monocytic cells exposed to seasonal and pandemic strains of influenza A viruses. Additionally, POP2 gene expression was found to be up-regulated in human donor PBMCs exposed to influenza A viruses. These findings support our notion that POP2 plays a critical role in ensuring a controlled, inflammatory response during human influenza A virus infection to favor protection over pathology.
The cysteinyl leukotriene receptor CysLTR1 mediates Th17 cell migration (CAM4P.156)
Authors: Kim Hyeong Su , Lee Gap
Published: January 2023

Th17 cells are one of CD4+ T cells that have crucial role in inflammation and autoimmunity. Although many studies revealed molecular mechanisms about Th17 cells but the migration of Th17 cells by lipid-based chemoattractant leukotriene signaling is not fully understood. Here, we show that Th17 cells are highly sensitive to leukotriene B4 and D4. Th17 cells expressed high levels of leukotriene B4 receptors and cysteinyl leukotriene receptors compare to other effector CD4+ T cell subsets. In chemotaxis assay, we confirmed that migration of Th17 cells was more efficient than that of other effector T CD4+ T cell subsets under the guidance of leukotriene B4 and D4, and it was blocked by specific inhibitors of leukotriene B4 receptor and cysteinyl leukotriene receptor. Finally in an animal disease model of experimental autoimmune encephalomyelitis (EAE), treatment with montelukast which is an inhibitor of cysteinyl leukotriene receptor, it mitigated clinical score of EAE. Thus, we concluded that leukotrienes act as chemoattractant for Th17 cells.
Antibody blocking of CLEC12A delays the course and attenuates EAE severity by impairing myeloid cell CNS infiltration: A potential new therapy for multiple sclerosis (CAM4P.158)
Authors: Sagar Divya , Singh Narendra , Huang Xiaofang , Ginwala Rashida , Philip Ramila , Nagarkatti Mitzi , Nagarkatti Prakash , Khan Zafar , Jain Pooja
Published: January 2023

The mechanism of dendritic cell (DC) recruitment across the blood brain barrier during neuroinflammation has been the least explored amongst all leukocytes. For cells of myeloid origin, while integrins function at the level of adhesion, the importance of lectins remains unknown. We pursued the study of a highly specific aspect of DC biology, namely the expression of surface C-type lectin receptors (CLRs) for their importance in adherence and transmigration across the blood-brain barrier in response to CCL2 chemotaxis. CLEC12A, an immunoreceptor tyrosine-based inhibitory motif (ITIM) known to recruit Src homology region 2 domain-containing phosphatase (SHP)-1/2, was subsequently identified as one such a target on DCs. SHP-1/2 phosphorylation was further found to facilitate the actin polymerization on DCs that express the WASP Interacting Protein (WIP). To test function of CLEC12A in an animal model of multiple sclerosis (MS), we administered an antibody to block CLEC12A that significantly ameliorated the progression and relapse of experimental autoimmune encephalomyelitis (EAE) in mice through inhibition of demyelination and myeloid cell infiltration into the CNS tissue. Correspondingly, a restoration of DC numbers in the periphery along with decrease in TH17 phenotype within CD4+ T cells was also seen. These studies reveal the utility of a DC-specific mechanism in designing new therapeutics for MS.