bluuebunny/crossref_metadata_2025 huggingface dataset:
Migration within chemoattractant-defined niches regulates B cell receptor signaling, lymphocyte differentiation and survival (P5087)
Authors: Pereira Joao , Beck Thomas , Gomes Ana
Published: January 2023

A critical feature of B and T lymphocytes is their ability to recirculate between lymphoid organs. Cell motility, besides catalyzing encounters between rare lymphocytes and cognate antigens, is also believed to promote lymphocyte survival. For B lymphocytes, however, little is known about how they migrate, and neither it is known if and how motility is required for survival. In previous studies, we demonstrated that B cells are already motile during early stages of development in bone marrow. Using intravital microscopy, we now demonstrate that B cell motility within bone marrow is controlled by continuous CXCR4 signaling and by α4β1 integrin mediated adhesion to VCAM-1, a surprising and unprecedented example of haptokinetic motility within lymphoid organ interstitium. Remarkably, B lymphocytes engineered to express pertussis toxin (PTX, B-PTX cells), an irreversible inhibitor of gαi protein coupled receptors (GPCRs) and of lymphocyte migration, displayed exacerbated tonic BCR signaling. Consequently, sessile B-PTX cells were unable to differentiate and survive past the transitional T2 stage in the spleen, which resulted in a ~10-fold reduced mature B cell compartment. Furthermore, engagement of GPCRs by chemoattractants in B-lineage cells selectively reduced Igα phosphorylation via a mechanism that involves kinase inhibition. In conclusion, our studies demonstrate an essential role for chemotaxis in B lymphocyte survival via a direct link between GPCR and BCR signaling.
α-Iso-cubebenol has therapeutic effect against sepsis via CXCR2 (P4203)
Authors: Lee Sung Kyun , Jung Young Su , Kim Sang Doo , Kook Minsoo , Lee Ha Young , Choi Young Whan , Bae Yoe-Sik
Published: January 2023

α-Iso-cubebenol, a natural compound isolated from the Schisandra chinensis fruit, strongly enhances survival rate in cecal ligation and puncture (CLP) challenge-induced sepsis. Mechanistically, α-iso-cubebenol markedly reduces viable bacteria in the peritoneal fluid and peripheral blood, by increasing production of superoxide anion. α-Iso-cubebenol also significantly attenuates widespread immune cell apoptosis in a mouse CLP sepsis model, and inhibits the production of proinflammatory cytokines including interleukin-1β (IL-1β) and IL-6 in CLP mice and lipopolysaccharide-stimulated splenocytes. We also found that α-iso-cubebenol stimulated calcium increase and degranulation in human neutrophils. α-Iso-cubebenol also strongly induced neutrophil chemotaxis, which was completely blocked by a CXCR2 antagonist, SB225002. The increased survival rateby α-iso-cubebenol was also significantly attenuated by SB225002. Taken together, the results indicate that α-iso-cubebenol-induced anti-septicactivity was mediated by CXCR2, suggesting CXCR2 as an important target for theregulation of sepsis and inflammation.
Salmonella Typhi survives in the host by evading a complement-dependent neutrophil immuno-surveillance (P4221)
Authors: Wangdi Tamding , Lee Cheng-Yuk , Spees Alanna , Yu Chen zhou , Heinrich Volkmar , Baumler Andreas
Published: January 2023

Vi capsular polysaccharide in S. Typhi is an important virulence factor. However, its role is incompletely understood. Previously, we have shown that the Vi capsule prevents complement component 3 mediated clearance of S. Typhi by preventing C3 fixation on the bacterial surface. Here we used isolated mouse and human neutrophils to show, at single cell level, that Vi negative S. Typhi mutant (tviB-vexE) directly triggers a chemotactic response by neutrophils when co-incubated in a medium containing autologous serum whereas Vi positive wild-type S. Typhi (Ty2) does not. Using neutrophils from C3 and C5aR deficient mice, we also show that this chemotactic response requires the complement component 5a receptor (C5aR) in neutrophils suggesting that the detection of generated C5a as a result of complement activation on the bacterial surface is a non-redundant signal used by neutrophils to move towards the bacteria. Additionally, data from our in vivo mouse model show that higher number neutrophils were associated with tviB-vexE than the WT S. Typhi an hour post interperitoneal injection of bacteria. Taken together, our data suggest that Vi capsule helps evade neutrophil detection of S. Typhi by preventing activation of C5a-dependent chemotactic response.
RvE1 and phagocytosis of Porphyromonas gingivalis by PMN in type 2 diabetes (P4227)
Authors: Van Dyke Thomas , Herrera Bruno , Hasturk Hatice , Kantarci Alpdogan
Published: January 2023

Periodontitis is a complication of Type 2 diabetes that impacts control of glycemia and systemic inflammation. Resolution of inflammation is an active, temporally orchestrated process of the biosynthesis of novel mediators (Resolvin E1, RvE1). Dysregulation of resolution may impact human inflammatory diseases such as diabetes and periodontitis. We examined the role of RvE1 in Type 2 diabetes and periodontitis by over-expressing the RvE1 receptor CMKLR1 in Type 2 diabetic mice (db-/-/db-/-); activation pathways and PMN phagocytosis of a periodontal pathogen Porphyromonas gingivalis (P.g.) were compared to wild type (WT), db-/-/db-/- and CMKLR1 over-expressing non-diabetic FVB WT. We report increased progression of periodontitis in diabetic mice is accompanied by impaired PMN phagocytosis. RvE1 enhanced phagocytosis of P.g. by WT PMN, but not PMN from diabetic animals. Phagocytosis was further increased by CMKLR1tg PMN. RvE1 rescued phagocytosis by db-/-/CMKLR1tg PMN. RvE1 induced reductions in AKT and MAPK phosphorylation that were enhanced in the transgenic animals. We conclude that PMN phagocytosis is impaired in db-/-/db-/- mice that is refractory to RvE1. RvE1 enhances PMN phagocytosis in non-diabetic WT mice and rescues impaired PMN phagocytosis in type-2 diabetic mice that over-express CMKLR1. Persistent infection and chronic inflammation characteristic of type 2 diabetes and periodontitis are due, in part, to failure of resolution of inflammation pathways.
Functional heterogeneity of monocyte populations during influenza virus infection (P4243)
Authors: Duan Susu , Sharma Shalini , Thomas Oguin , Morris Melissa , Thomas Paul
Published: January 2023

Influenza infection in mice is characterized by waves of recruitment of innate immune cells into the lung. We have previously described TNF-α/iNOS-producing monocytes (TipDC) as promoting the anti-influenza adaptive immune response and contributing to disease pathogenesis. Here, we report the identification of a novel subset of monocytes, distinct from TipDCs and lung resident innate populations, that is recruited into the lung during influenza infection in mice. These monocytes had distinct morphology, phenotypic lineage marker expression (CD8α, CD103, F4/80 and Ly6C etc) and transcriptional profile from TipDCs. CCR2 signaling contributed to the recruitment of this monocyte subset into the lung, but it was not indispensable. They were not found in the lung-draining lymph nodes. Functionally, both these monocytes and TipDC could not stimulate CD4 T cell proliferation, but only this subset of monocytes suppressed CD4 and CD8 T cell proliferation, and they also induced high levels of Th17-associated cytokines from CD4 T cells. Our results highlight the heterogeneity of monocyte populations and their functional roles in anti-viral immunity to influenza infection. This heterogeneity may be harnessed for immune modulation strategies as novel therapeutics for influenza infection.
Mast cell mediated immune pathology during dengue virus infection (P4245)
Authors: St. John Ashley , Rathore Abhay , Raghavan Bhuvanakantham , Ng Mah Lee , Soman Abraham
Published: January 2023

Mast cells (MCs) promote immunosurveillance for pathogens in peripheral tissues and line the host vasculature. We observe that MC are activated by dengue virus (DENV), a flavivirus that is spread by mosquito vectors. MC activation by DENV during viremia elevates systemic levels of their vasoactive products, including the MC-specific protease, chymase, and promotes vascular leakage. Symptoms of DENV infection in humans include widespread vascular leakage, pooling of fluid within internal organs and, occasionally, severe hemorrhaging; however, the processes that underlie the immune-mediated vascular pathology during dengue infection are largely unknown. A component of dengue-induced vascular leakage is MC-dependent since mice that lack MCs have greatly reduced vascular permeability during infection. We show that treatment of infected animals with MC-stabilizing drugs restores vascular integrity during experimental DENV infection. Validation of these findings using human clinical samples revealed a direct correlation between MC activation and DENV disease severity based on the measurement of chymase as a serum biomarker. Our findings reveal that MCs mediate DENV-induced vascular pathology, and suggest MC-stabilizing drugs should be evaluated for their effectiveness in improving disease outcomes during viral hemorrhagic fevers.
Oxidation-specific epitopes are an important target of innate immunity (P4261)
Authors: Gonen Ayelet , Oliveira Romulo , Diehl Cody , Cole Jason , Copps Jeffrey , Nizet Victor , Ghosh Partho , Witztum Joseph
Published: January 2023

Oxidation of LDL is important in atherosclerosis and generates oxidation-specific epitopes, which are proinflammatory and immunogenic. We showed that oxidation specific epitopes are recognized by innate immunity, including Natural Ab’s, scavenger receptors and innate proteins. In addition, many of these epitopes are also found on apoptotic cells and are share molecular mimicry with pathogens. For example, the NAb E06, scavenger receptor CD36 and CRP, all bind to PC on oxidized LDL, but also bind to the PC on apoptotic cells and on S. pneumonia. To determine if other oxidation specific epitopes share molecular mimicry with pathogens, we screened a pathogen library with oxidation specific epitopes NAbs. We found that the NAb E014, specific for malondialdehyde also bound to Streptococcus group A, demonstrating molecular mimicry between the two. I showed that E014 specifically binds to the M protein of Streptococcus group A. Using synthetic peptide arrays I narrowed the binding site on M protein to a 24AA section. I am currently studying the immunological consequences of molecular mimicry between peptides from M protein and the ubiquitous malondialdehyde. These data support the hypothesis that oxidation-specific epitopes are an important target of innate immunity and both oxidative events and pathogens have contributed to the natural selection of shared and potent innate immune responses to oxidative specific epitopes.
The effects of HIV-1 Tat protein on mycobacterial infection (P4219)
Authors: Li James , Pong John , Lau Allan
Published: January 2023

Human immunodeficiency virus infection remains a worldwide threat. With the emergence of HIV-1 induced immunodeficiency in the host, mycobacterial infection becomes a common opportunistic infection as its prevalence is dramatic increased. HIV-1 Tat is an important viral regulatory protein in promoting HIV replication and modulating the cytokine production, as demonstrated by our previous publications. In light of that, we would like to investigate whether HIV-1 Tat could affect mycobacteria induced immune responses. We first examined whether HIV-1 Tat could mediate mycobacteria induced cytokine production. The results demonstrated that HIV-1 Tat suppressed mycobacteria-induced TNF-α in human primary macrophage. Next, we examined whether HIV-1 Tat also suppress the intracellular survival of mycobacteria. Under the pretreatment of HIV-1 Tat in human primary macrophages, intracellular growth of mycobacteria was enhanced significantly. Since TNF-α is proposed to regulate mycobacterial survival, we would like to verify this hypothesis in order to provide the evidence to support our findings that HIV-1 Tat downregulated mycobacteria induced TNF-α is participated in suppressing the mycobacterial survival. The results showed that when we neutralized the mycobacteria produced TNF-α by antibodies, the survival of mycobacteria is increased. In summary, HIV-1 Tat may play a role in favouring mycobacterial opportunistic infection during AIDS progression.
Peptidoglycan acts as a Gram-positive endotoxin (P4035)
Authors: Coggeshall K. , Raisley Brent , Silasi-Mansat Robert , Sun Dawei , Keshari Ravi , Popescu Narcis , Kurosawa Shinichiro , Lupu Florea
Published: January 2023

We proposed that peptidoglycan (PGN) contributes to the systemic inflammation and sepsis accompanying Gram-positive infections. However responses are dependent on pre-existing IgG recognizing peptidoglycan and capable of engaging Fcgamma receptors. Mice lack these antibodies and hence respond weakly to in vivo PGN challenges. Non-human primates possess anti-PGN IgG and have been a useful and informative model for sepsis, We challenged 4 baboons with varying doses (7-48 mg/Kg) of PGN and noted: vascular leakage, increased circulating proinflammatory cytokines, increased activation of neutrophils and platelets, expression of tissue factor on monocytes, massive consumption of clotting factors and complement proteins, and microthrombosis-induced kidney failure. The severity of the outcome correlated with the pre-existing titer of anti-PGN antibodies such that animals with a higher titer had a poorer outcome. The pathological features induced by the sterile PGN infusion greatly resemble the pathological features induced by infusion of live bacteria from which the PGN was generated. The data support our hypothesis that PGN and anti-PGN antibodies are a major contributor to Gram-positive sepsis.
β-Glucuronidase is a key regulator of murine Lyme arthritis severity and rheumatoid arthritis (P4013)
Authors: Bramwell Kenneth , Ma Ying , Weis John , Teuscher Cory , Zachary James , Weis Janis
Published: January 2023

Lyme Disease is the most prevalent arthropod borne illness in the U.S. Up to 60% of patients develop inflammatory arthritis following infection. Host genetics play a key role, as inbred mouse strains exhibit consistent differences in Lyme arthritis severity. Our lab previously described maximal linkage (LOD 10.2) of disease severity to the Bbaa2 locus on mouse Chromosome 5. Recently, we developed advanced congenic mice carrying sub-intervals of Bbaa2 from susceptible C3H on an otherwise uniform resistant B6 genetic background. This led to the positional cloning of a novel disease regulator, the lysosomal enzyme β-Glucuronidase (Gusb). C3H mice are Gusb hypomorphs, exhibiting a 90% enzymatic deficiency. Two sub-strains of CBA mice carrying the hypomorphic or wild type Gusb alleles develop severe Lyme arthritis or are protected, respectively. A Gusbnull mouse strain on the B6 background develops severe Lyme arthritis, while heterozygotes are protected. Transgenic overexpression of Gusb in C3H mice to correct the defect led to reduced arthritis severity, indicating a single gene effect. Preliminary data show that Gusb congenic mice also develop more severe disease in the KBxN model of Rheumatoid arthritis, suggesting a conserved role. Deficiencies in Gusb and other lysosomal enzymes lead to lysosomal storage diseases. We found that treatment with Hydroxychloroquine, which accumulates in lysosomes, dramatically reduces Lyme arthritis severity in Gusb congenic mice.
Anti-tumor responses driven by alloreactivity (P2058)
Authors: Fast Loren , Reagan John , Nevola Martha , Quesenberry Peter
Published: January 2023

Previous work by our group found that infusion of G-CSF mobilized haploidentical donor white blood cells at a dose of 1 - 2 x 108 CD3+ cells/kg into refractory cancer patients who had received 100 cGy total body irradiation resulted in responses in a majority (14/27, 9 major) of patients with hematological malignancies despite elimination of the donor cells. The effector mechanism(s) responsible for these anti-cancer responses were not determined although it was noted that there was a cytokine storm that appeared within a day following the infusion of cells. One possible mechanism is that the alloreactive response overrides inhibitory mechanisms already in place in the cancer bearing patient allowing cancer reactive effector cells that are already present to become activated and lyse cancer cells. To test if this is the case, T cells were isolated from peripheral blood mononuclear cells obtained from newly diagnosed leukemic patients, mixed with fully allogeneic stimulator cells (from 1 or 2 different donors) and then tested for their ability to proliferate in response to the allogeneic stimulator cells on day five and for their ability to lyse autologous leukemia cells after stimulation for seven days. Anti-leukemic effector cells could be generated in eight out of the sixteen patients enrolled to date. The characteristics of the effector cells and the explanations for the lack of responses in some patients are the subject of current ongoing studies.
IL35 neutralization enhances tumor specific immunity in mouse models of cancer (P2024)
Authors: Turnis Meghan , Gravano David , Delgoffe Greg , Steinkamp Heidi , Vignali Dario
Published: January 2023

Regulatory T cells (Treg) are critical to maintenance of self-tolerance and prevention of autoimmunity; however, they also function to limit anti-cancer responses.Therefore, clarifying the mechanisms of Treg-mediated suppression in the context of cancer will aid in the rational design of new therapies.Our lab has identified a novel suppressive role for the IL35 heterodimer in Treg. IL35 is a member of the IL12 family composed of IL12a and Ebi3 subunits and is preferentially expressed by Treg. Treg and other suppressive factors in the tumor microenvironment inhibit antigen-specific cytotoxic lymphocyte responses; depletion or inhibition of Treg has been shown to decrease tumor burden and enhance patient outcome in both animal models and the clinic. We hypothesize that IL35 contributes critically to the tumor microenvironment and that its elimination will enhance tumor specific immunity. Using neutralizing antibodies specific for IL35-Ebi3, we have shown that systemic neutralization of IL35 significantly slows tumor growth compared with IgG control and enhances survival in the B16 melanoma model. Molecular analysis of tumor-infiltrating lymphocytes (TILs) has shown increased numbers, more activated CD8+ cells (CD44+CD62L-), and a greater proportion of proliferative cells by BrdU/Ki-67 staining in IL35-neutralized animals. We are currently extending studies to include metastatic and genetic models to better understand the mechanism of IL35 activity in the tumor microenvironment.
A novel CXCR7-targeted scFv-FC chimeric antibody kills CXCR7+ glioblastoma and leukemia cancer cells by antibody dependent cellular cytotoxicity (P2060)
Authors: Carlson Jeffrey , Jang Andrew , Burke Thomas , Lewen Susanna , Huang Kexin , Tu Hua , Zabel Brian
Published: January 2023

CXCR7 is an atypical chemokine receptor that plays a critical role in guiding progenitor cell migration during embryo- and organo-genesis. Following fetal development and birth, however, CXCR7 protein is difficult to detect on cells or tissues except in the context of cancer. CXCR7 is upregulated by a number of primary tumor types, including human B cell lymphoblasts and glioblastomas, and is selectively expressed on vascular endothelial cells associated with solid tumors. We engineered a chimeric single chain Fv-human FC (IgG1) antibody (X7scFv-huFC) that selectively targets and kills CXCR7+ human B cell leukemia and glioblastoma cell lines by antibody dependent cellular cytotoxicity (ADCC). Safety and pharmacokinetic studies of X7scFv-huFC in rodents will be presented, and our progress in efficacy studies, including xenograft models of human leukemia and glioblastoma in SCID mice, will be discussed.
Global targeting of MDSC escape mechanisms cures advanced 4T1 breast tumors (P2062)
Authors: Zorro Manrique Soraya , Spencer Christopher , Dominguez Ana , Bradley Judy , Mirza Noweeda , Ko Jennifer , Viso Carole , Gades Naomi , Finke James , Pease Larry , Gendler Sandra , Cohen Peter
Published: January 2023

4T1 is a metastatic breast cancer model with fulminant accumulation of MDSCs. Survival and function of MDSCs is often reported as STAT3-dependent, and STAT3 inhibitors such as sunitinib dramatically deplete 4T1 splenic MDSCs. Paradoxically, sunitinib fails to eradicate 4T1 intratumoral MDSCs or prevent tumor progression. We hypothesized that STAT3 activation is a consistent feature of splenic but not intratumoral MDSCs. PhosphoSTAT analyses of 4T1-bearers confirmed that splenic MDSCs of all Gr1 intensities were solely pSTAT3pos. In remarkable contrast, intratumoral Gr1high MDSCs largely lacked any pSTAT activation, and intratumoral Gr1dim MDSC precursors displayed varied expression of pSTAT5 and pSTAT1 in addition to pSTAT3. We therefore sought global means to target MDSCs. We determined that 4T1 and other tumor models could be cured by repetitive administration of cyclophosphamide alternating with TLR agonists such as CpG ODN1826. While cure required participation of host CD4+ and CD8+ T cells, global targeting of MDSCs was also observed: (1) as with sunitinib, pSTAT3pos splenic MDSCs were eradicated; (2) pSTAT expression by intratumoral MDSCs was fully abolished; (3) the remaining intratumoral MDSCs, all pSTATneg, outsurvived all other cells, and were likely induced to serve as the final mediators of tumor rejection. In conclusion, strategies which globally target MDSCs and promote the endogenous anti-tumor T cell response can cure advanced metastatic tumors.
Plasmacytoid and CD8α DC are both necessary to generate antitumor immunity following Ad5-TRAIL/CpG immunotherapy (P2078)
Authors: James Britnie , Kucaba Tamara , Griffith Thomas
Published: January 2023

It is now appreciated that there are distinct subsets of dendritic cells (DC) with specialized functions. Plasmacytoid DC (pDC) and CD8α DC can contribute to the priming, activation, and function of antitumor CD8 T cells; however, their specific roles and necessity in stimulating antitumor immunity is not clearly understood. Thus, we examined the distinct role pDC and CD8α DC play in an orthotopic model of metastatic renal cell carcinoma (RCC) treated with a recombinant adenovirus encoding TRAIL (Ad-TRAIL) combined with CpG ODN. Wild-type (WT) mice bearing RCC tumors and treated with Ad-TRAIL/CpG ODN cleared primary and metastatic tumors, and these mice survived long-term. In contrast, mice specifically depleted of either pDC (using an α-PDCA1 mAb) or CD8α DC (Batf -/- mice) had uncontrolled tumor growth and high mortality regardless of treatment. Both pDC-depleted and Batf3-/- mice exhibited significantly decreased infiltration of effector CD8 T cells in the primary tumor site, as compared to WT mice after therapy, possibly related to the reduced expression of pro-inflammatory cytokines in pDC-depleted mice and reduced uptake of apoptotic tumor cells in Batf3-/- mice. These data collectively suggest that both pDC and CD8αDC carry out independent, but complementary roles that are necessary to initiate an efficacious antitumor immune after Ad5-TRAIL/CpG ODN therapy.
Contribution of CCDC134, a potential cytokine, inducing CD8+ T cell-dependent antitumor effect in cancer immunotherapy (P2086)
Authors: Xiao Lin , Shao Wenwei , Gong Xiaoting , Huang Jing , Qiu Xiaoyan
Published: January 2023

CCDC134 (coiled-coil domain containing 134) was firstly identified to be a novel classical secretory protein by our lab. Although we previously identified CCDC134 as a novel MAPK-regulator which down-regulated the Raf-1/MEK/ERK and JNK/ SAPK pathways in some cancer cell lines. However, the critical function of CCDC134 still remains unclear. In view of CCDC134 characteristic, which was widely expressed in lymphocytes, especially up-regulated expression in activated CD4+, CD8+ T lymphocytes and maturated DC, we proposed the hypothesis if CCDC134 was involved in immune response as a potential cytokine. Here, we first found that CCDC134 greatly promoted activation, proliferation and cytotoxicity of CD8+ T cells via augmenting the expression of activation molecules (CD25, CD69) and effector molecules, such as IFN-γ, TNF-α, granzyme B and perforin in vitro. Moreover, by a series of experimental evidences, we have found that CCDC134 displayed strong anti-tumor activity in a CD8+ T cells, but not CD4+ T cells, and IFN-γ-dependent manner in vivo. In addition, CCDC134 markedly promoted phosphorylation of STAT5, but not STAT1, STAT3 in CD8+ T cells and CD8+ T cells proliferation augmented by CCDC134 was dependent on STAT5. Our data collectively suggested that CCDC134, perhaps was served as a so far unidentified cytokine-like protein, which showed strong anti-tumor effects via augmenting CD8+ T cells-mediated tumor immunity.
Measuring potency of bispecific effector/target-linking antibodies for inducing cytotoxicity of tumor cells using an impedance-based assay platform (P2094)
Authors: Young Judy , Montgomery Harley , Sun Laura , Junttila Teemu , Mai Elaine
Published: January 2023

Cancer patients mount weak tumor-specific T-cell responses due to immune escape mechanisms of tumors. Although cytotoxic T-cells are potent killers, activation requires antigen processing, specific recognition, and co-stimulation. Bispecific antibody-derived proteins that bind both CD3 and tumor antigens can bypass these requirements by directly linking cytotoxic T cells to tumor cells and have been shown clinically to enhance T cell-mediated tumor cell killing. Our goal was to assess the relative potency of anti-CD3/anti-tumor antigen full-length IgG1 bispecific antibodies. Assays based on electrical impedance are well suited for tumor cell killing assays because the adherent target cells generate an impedance signal, while the non-adherent effector cells do not. Human tumor cells were plated with CD8+ T cells and bispecific antibodies, and cytotoxicity was recorded in real time at intervals during culture. Tumor cell killing was found to be dependent on the bispecific antibody concentration, with EC50 values of 27-40 pM. In addition, killing was dependent on the number of CD8 T cells added, with an optimal effector:target cell ratio of 4:1. Compared to a well established but labor-intensive FACS-based assay performed in parallel, the results of the impedance assay agreed closely. Moreover, impedance-based cell killing assays have the advantages of real-time, label-free readings as well as simple assay development and performance.
CD4+ T cell-mediated protection against tumors: no bystander killing (P2114)
Authors: Schjesvold Fredrik , Tveita Anders , Sundnes Olav , Haraldsen Guttorm , Bogen Bjarne
Published: January 2023

Background: MHC class II-restricted CD4+ T cells reactive against the Id peptide of MHC class II-negative MOPC315 murine myeloma cells can protect mice against injected tumors. This protection occurs via indirect recognition of secreted tumor-specific antigen, presented on MHC class II-molecules of tumor-infiltrating CD11b+ cells. Activated Id-specific Th1 cells produce IFNγ that activate macrophages which become cytotoxic to tumor cells. We wanted to assay the ability of such CD4+/M1 macrophage mediated protection to prevent the outgrowth of antigen-loss tumor variants. Methods: Id-specific TCR-transgenic SCID mice were challenged with a mixed population of Id-secreting and non-secreting MOPC315 cells, differentially labeled with fluorescent proteins. Tumor growth was monitored by in vivo imaging. Tumor-infiltrating macrophages were assayed for their ability to inhibit tumor growth. Results: In the presence of a large excess of antigen-secreting tumor cells, outgrowth of antigen loss-variants occurred with no evidence of bystander killing. A predominance of alternatively activated macrophages was seen in areas dominated by antigen loss-variants, whereas classic activation was the dominant phenotype in areas populated by regressing Id-secreting tumor cells. In vitro, growth of antigen loss-variants was completely inhibited by the provision of the tumor-specific antigen. This data suggests strict spatial requirements of antigen for effective macrophage-mediated protection.
Lenalidomide increases peripheral T regulatory phenotype cells in patients with HIV associated Kaposi Sarcoma (P2030)
Authors: Han Semi , Bui Jack , Reid Erin , Arora Sumit
Published: January 2023

Kaposi Sarcoma, an angioproliferative vascular tumor, is the most common malignancy in patients with HIV and presents a treatment conundrum because of its variable natural history. Lenalidomide is a drug that has immune-modulatory, anti-neoplastic, and anti-angiogenic properties. It is used in the treatment of myelodysplastic disease and multiple myeloma, but its in vivo effects on peripheral blood immune activity is not fully explored. In this preliminary report from a phase I/II trial of lenalidomide in the treatment of HIV associated Kaposi Sarcoma, we found that lenalidomide increased the number and percentage of T regulatory phenotype cells in the peripheral blood. In patients with measurable regression of Kaposi Sarcoma lesion size as well as number of lesions, there was also an observable accumulation in FOXP3 expressing cells in the Kaposi Sarcoma lesion. These findings enable us to speculate on three novel concepts: 1) lenalidomide increases the frequency of circulating T regulatory phenotype cells, 2) lenalidomide’s anti-angiogenic mechanism may be due to T regulatory cell induction, and 3) in contrast to their role in promoting tumor growth by inhibiting tumor immunosurveillance, T regulatory cells may inhibit angioproliferative tumors therefore act as an effective therapeutic agent for tumors such as Kaposi Sarcoma.
OSU-2S: a novel non-immunosuppressive FTY720 derivative mediates potent cytotoxic activity through PKC dependent phosphorylation of tumor suppressor SHP1 and down modulation of Tcl1 oncogene in chronic lymphocytic leukemia (P2120)
Authors: Mani Rajeswaran , Frissora Frank , Mao Yicheng , Chiang Chi-ling , Mo Xiaokui , Jarjoura David , Baskar Sivasubramanian , Rader Christoph , Lee Robert , Lee L.James , Chen Ching-Shih , Byrd John , Muthusamy Natarajan
Published: January 2023

We have recently demonstrated cytotoxic activity of FTY720, a sphingosine analogue, against several B cell malignancies including chronic lymphocytic leukemia (CLL).To overcome the limitations associated with immunosuppressive properties of FTY720, we have developed OSU-2S, a synthetic FTY720 derivative with cytotoxic potency and negated immunosuppressive property. Treatment of primary CLL cells with OSU-2S resulted in potent cytotoxicity associated with PKC mediated phosphorylation of Serine591 (S591) of SHP1 and its nuclear translocation consistent with a potential role for S591 phosphorylation. Concentrations of bisindolylmaleimide that inhibited PKC and thus SHP1S591 phosphorylation abrogated OSU-2S mediated cytotoxicity in CLL cells. Gene expression profiling (GEP) revealed OSU-2S mediated down regulation of TCL1A. Consistent with this OSU-2S induced down regulation of TCL1A at mRNA and protein levels in primary CLL cells. The therapeutic efficacy of OSU-2S was demonstrated in Eµ-Tcl1 transgenic CLL mouse model and SCID-Raji xenograft models with significant reduction in leukemic B cells. Further, administration of OSU-2S immunoliposome targeting receptor tyrosine kinase-like orphan receptor (ROR1) expressed in lymphoid malignancies including CLL in Eμ-human ROR1 transgenic mice resulted in selective depletion of ROR1 positive B cells. In summary, we have identified OSU-2S as a modulator of tumor suppressor and oncogene targets in CLL resulting in potent cytotoxicity.
Kinetics of MOG-specific CD4+ T cells in experimental autoimmune encephalomyelitis. (P4127)
Authors: Martinez Ryan , Blanchfield Jennifer , Evavold Brian
Published: January 2023

During experimental autoimmune encephalomyelitis (EAE), effector CD4+ T cells are primed in the periphery and migrate to the central nervous system (CNS) to mediate immune damage to myelin. In experiments described here, we investigated the myelin oligodendrocyte glycoprotein (MOG)-specific CD4+ T cell immune response during C57BL/6 EAE before disease onset. The peripheral lymphoid organs, blood and CNS were analyzed using MHC class II tetramer enrichment to determine the precursor frequency, kinetics, and phenotype of MOG-specific CD4+ T cells during priming and migration to the CNS. We found that naïve B6 mice had a precursor frequency of about 450 MOG-specific CD4+ T cells in peripheral lymphoid organs, which expanded to ~30,000 at day 21 after EAE induction. Our lab has previously shown that identification of myelin-specific CD4+ T cells in the CNS is underestimated using MHC class II tetramers, so we attempted to determine if there were more low affinity MOG-specific CD4+ T cells measureable by the sensitive micropipette adhesion frequency assay. We found, that similar to the CNS, MHC class II tetramer enrichment underestimated the amount of low affinity MOG-specific CD4+ T cells in the peripheral lymphoid organs when compared to micropipette binding frequency assay. These results demonstrate that low affinity, self-antigen specific CD4+ T cells are at higher frequency than previously appreciated, and may play an important role in the pathogenesis of EAE.
The role of TNFα pathway and microRNA in regulating autoimmune regulator gene expression and translation (P4101)
Authors: Nagafuchi Seiho , Oda Yoshie , Matsuo Tomohito , Kurisaki Hironori , Katsuta Hitoshi , Nagafuchi Seiho
Published: January 2023

Autoimmune regulator (AIRE) is a responsible gene for autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), presenting multiple autoimmune diseases. AIRE gene is expressed in the thymus and lymph nodes. Thus, AIRE gene plays an important role in both the central and peripheral regulation of immune responses. However, there are some patients with APECED, who lack mutations in AIRE gene. Therefore, dysfunction of the regulatory mechanism of AIRE gene may be associated with the pathogenesis of APECED. In this study, we found that nuclear factor-κB (NF-κB) pathway regulated AIRE gene expression during maturation process of dendritic cells under TNFα stimulation, along with the significant elevation of AIRE gene expression. By inhibiting NF-κB pathway, AIRE gene expression was decreased. We also found that miR-220b inhibited the translation of AIRE gene, binding 3’ untranslated region (3’UTR) of the target mRNA. AIRE protein expression was inhibited by overexpressing microRNAs in 293T cells transfected with AIRE gene which have 3’UTR plasmid but not in AIRE open reading frame (ORF) alone. At least, miR-220b was not expressed in the normal thymus which expresses high level of both AIRE gene and AIRE protein, to delete autoreactive T cells. Thus, it was concluded that there are multiple the regulatory mechanisms of AIRE gene expression, and deterioration of those mechanisms may be associated with APECED phenotype.
Critical role of Annexin A1 in protection against pulmonary Mycobacterium tuberculosis infection. (P3320)
Authors: Tzelepis Fanny , Gillard Joshua , Jaworska Joanna , Nishimura Tomoyasu , Verway Mark , Hassani-Ardakani Kymia , Remold Heinz , Vali Hojattolah , Maziar Divangahi
Published: January 2023

Annexin1 is a 37kDa protein with wide spectrum of biological activity. Although there are data emphasizing on anti-inflammatory role of Annexin1 in inflammatory diseases, little is known about its role in pathogenesis of tuberculosis. Recently, it has been described the involvement of Annexin1 in the formation of apoptotic envelope, generation of “eat me” signal and activation of Tcells. As phagocytosis of apoptotic vesicles from Mycobacterium tuberculosis (Mtb) infected macrophages is one of the main sources of antigens for CD8T cells via cross-presentation, we hypothesized that impaired capacity of Annexin1-/-DC in cross-presentation increases host susceptibility to Mtb infection. We found that Annexin1-/-mice were severely susceptible to Mtb infection. High levels of pulmonary bacterial burden and mortality in Annexin1-/-mice were associated with reduced antigen-specific CD8T cell response in the lungs. This reduced response was not due to an intrinsic role of Annexin1 in macrophage apoptosis or T cell activation as generation of chimeric mice in which only T cells were deficient in Annexin1 did not show impairment in Tcell mediated immunity or protection against Mtb infection. Interestingly, both in vitro and in vivo, Annexin1-/-DC demonstrated a significantly reduced capacity to cross-present antigens to CD8Tcells. Together, these data identify Annexin1 as a central player in protective immunity against Mtb infection, by regulating the power of DC in cross-presentation.
IL-10 from T cells, but not macrophages is required for chronic disease in Leishmania mexicana infection (P3328)
Authors: Buxbaum Laurence
Published: January 2023

Chronic cutaneous disease of mice caused by the protozoan parasite Leishmania mexicana requires IL-10 and FcγRIII (an activating IgG receptor). Macrophages are abundant in lesions and readily secrete IL-10 in response to IgG-coated amastigote stage parasites, making macrophages a prime candidate as the critical source of IL-10. However, both direct and indirect evidence demonstrated that macrophage IL-10 is not essential for chronic disease. Specifically, macrophage-granulocyte specific IL-10-deficient mice have chronic disease identical to wild-type mice. We now show that T cell-derived IL-10 is required for chronic disease. CD4-cre IL-10flox/flox mice lack IL-10 from T cells (both CD4+ and CD8+) and heal their L. mexicana lesions. We had previously shown that depletion of CD25+ T cells had no effect on chronic disease, and thus T cells other than Treg cells should be the important source of IL-10. Given that conventional T cells do not express FcγRs, there is likely to be an indirect pathway by which FcγRIII engagement on some other cell induces IL-10 from T cells. Further work is needed to delineate these pathways.
T cell cytokine profile of early tuberculosis infection and disease in Cynomolgus macaque model (P3335)
Authors: Gideon Hannah , Lin Philana , Flynn JoAnne
Published: January 2023

Mycobacterium tuberculosis (Mtb) remains one of the world’s most successful human pathogen. Addressing questions on immunological events soon after Mtb infection in human is challenging. Non-human primate model (Cynomolgus macaques) provide the necessary platform to understand the early immunological events in Mtb infection. We infected 28 Cynomolgus macaques with a low dose of Mtb (Erdman strain, ~25-50 CFU) and monitored clinically for signs and symptoms of tuberculosis up to six months post infection and classified to have either latent TB infection or active disease based on chest X-ray and Mtb culture. Peripheral blood was collected at time-points: pre Mtb infection; and at days: 10, 20, 30, 42, 56, 90, 120, 150 and 180 post infection. PBMC were isolated and multi-parametric intracellular flow cytometry was performed in fresh PBMC to track the early CD4 and CD8 T cell cytokine profiles (CD107a, GMCSF, IFN-g, IL-2, IL-4, IL-6, IL-10 and IL-17) during the course of Mtb infection. Preliminary analysis shows difference in the cytokine response in the early phase of infection between those who developed active disease or latent infection. In addition, we find a spectrum of cytokine response, which overlap between active and latent tuberculosis.
Intrinsic role of MyD88 for anti-fungal Tc17 cell responses (P3338)
Authors: Nanjappa Som , Wuthrich Marcel , Klein Bruce
Published: January 2023

Our previous studies showed that vaccine-induced Tc17 cells are necessary for antifungal defenses to control lethal pneumonia due to experimental blastomycosis. Although dendritic cell cytokines such as IL-6, TGF-β and IL-1 are generally required for differentiation of Th17 cells, the role of intrinsic MyD88 signaling and upstream pattern recognition receptors (PRRs) on CD8 T cells for anti-fungal Tc17 responses has not been studied. In work here, we found that MyD88 expression in CD8 T cells was essential for the generation of Tc17 responses following vaccination. As IL-18, IL-1 and PRRs mediate signaling through MyD88, we investigated the requirement of each of these components for anti-fungal Tc17 responses. We found in vivo that IL-18R was dispensable and that IL-1R1 signaling was insufficient to account for the Tc17 defect observed in MyD88-/- CD8 T cells, suggesting a potential role for PRRs. Our in vitro data also suggested that TLR2 signaling is required for Tc17 differentiation. Interestingly, Tc1 responses were relatively spared in MyD88-/- CD8 T cells. Mechanistically, MyD88-/- Tc17 not Tc1 cells failed to proliferate and levels of Bcl2 and Bcl-xL were unaffected, indicating poor expansion but not death of MyD88-/- Tc17 cells as a cause of poor Tc17 responses. Thus, intrinsic MyD88 and PRR signals in CD8 T cells are crucial for the generation of anti-fungal Tc17 responses. These data may have potential applications for designing fungal vaccines.
IL-12 regulated checkpoints in the CD8+ T cell response to Toxoplasma gondii (P3340)
Authors: Shah Suhagi , Grotenbreg Gijsbert , Rivera Amariliz , Yap George
Published: January 2023

We have previously shown that IL-12 is necessary for the differentiation of KLRG1+ IFN-γ producing CD8+ T effector cells during the immune response to Toxoplasma gondii. However, which key events during the activation, differentiation and mobilization of CD8+ T effector cells are critically regulated by IL-12 have not been identified. By tracking the dynamics of endogenous and adoptively transferred T. gondii-reactive CD8+ T cells, we were able to identify two key IL-12 dependent checkpoints. IL-12 is critical for the early diversification of CD8+ T cells into KLRG1+ IFN-γ-producing T effector cells, which occurs even prior to their initial proliferation. Unexpectedly, acquisition KLGR1 expression was accompanied by an attenuation of CXCR3 expression and this down regulation was IL-12 dependent. We postulate that this IL-12-driven reduction of CXCR3 expression may allow these mature KLRG1+ CD8+ T cells to outmigrate from secondary lymphoid organs. Failure to suppress CXCR3 may result in the retention of KLRG1+ CD8+ T effector cells in secondary lymphoid organs and underlie the deficit in effector cell numbers observed at the site of T. gondii infection in IL-12 p35-/- hosts. Our study reveals that IL-12 implements the early diversification of CTLs by both promoting and attenuating expression of key CD8+ T lymphocyte genes required for their effector function and mobilization.
BCG-treated M2-macrophages enhance IFN-γ production of T cells (P3348)
Authors: Lardone Ricardo , Guo Yi , Plaisier Seema , Singh Babu , Teles Rosane , Modlin Robert , Sieling Peter , Lee Delphine
Published: January 2023

M1- and M2-polarized Macrophage (MΦ) subtypes differentially regulate anti-mycobacterial defense. To examine their roles in human infection, we compared the gene expression profile of M1- vs. M2-MΦ to the expression profile of lesional skin from patients with restricted (tuberculoid, T-lep) vs. disseminated (lepromatous, L-lep) leprosy (a mycobacterial skin infection). Rank-Rank Hypergeometric Overlap (RRHO) is a novel algorithm for comparing full gene expression profiles from two independent microarray experiments that generates the most statistically significant overlapping gene list without requiring strict p-value/fold change cutoffs. RRHO analysis showed significant overlap of genes between M2-MΦ and L-lep lesions (n = 1559, max log hypergeometric p-value = 196), indicating a role for M2-MΦ in immune unresponsiveness. Bacillus Calmette-Guerin (BCG) is given to protect household contacts against leprosy. We hypothesized that BCG treatment may affect the function of M2-MΦ and investigated its effects in vitro. We found that BCG-treated M2-MΦ secreted less IL-10 (p < 0.05), with no effect on IL-12p40, compared to untreated M2-MΦ. In addition, BCG-treated M2-MΦ increased the frequency of IFN-γ producing cells in an antigen non-specific manner (p < 0.05), suggesting an adjuvant effect of BCG. Our data indicates M2-MΦ can be reprogrammed by BCG, resulting in increased IFN-γ production to enhance anti-mycobacterial immunity.
Modeling immune receptor clustering with nano-scale discoidal membrane bilayers (P3350)
Authors: Anikeeva Nadia , Sykulev Yuri , Blanchette Creig , Fisher Nick
Published: January 2023

MHC proteins on antigen-presenting cells form clusters with each other and other cell surface ligands. The changes in the clustering serve as a sensitive mechanism to modulate T cell responses. We propose to mimic MHC membrane patches using nanolipoprotein particles (NLPs). NLPs are nano-scale discoidal lipid bilayers stabilized by apolipoproteins. Nickel-chelating lipids incorporated into the NLP bilayer capture soluble polyhistidine-tagged pMHC. We have studied the interactions of cognate (non-self) and non-cognate (self) pMHC/NLPs with CD8+ CTL. Both self and non-self pMHC/NLPs were bound to the T-cell surface but to different extents. The apparent equilibrium binding constants of both non-self and self pMHC/NLPs were strongly dependent on pMHC density. A 3-fold difference in pMHC density (from 10:1 to 30:1 per NLP) caused 10-fold and 30-fold increases in the apparent affinity, respectively. Surprisingly, the avidity of self and non-self multivalent interactions appears to be similar when the pMHC density on the NLP surface is high. The similar avidity may be explained by a very rapid on-rate of CD8 binding to clustered MHC and marginal difference in the dissociation rates at the high epitope density resulting from very efficient CD8-MHC rebinding. NLP-based model MHC clusters with pre-determined properties could be incorporated onto glass beads coated with a lipid bilayer to engineer artificial APC with the goal of inducing ex vivo long-lived T-cell effectors for adoptive T-cell therapy.
A new and robust method of tethering IgG surrogate antigens on lipid bilayer membranes to facilitate the TIRFM based live cell and single molecule imaging experiments (P3352)
Authors: Liu Wanli , Zhang Shaosen , Xu Liling , Zhao Xingwang , Xu Yinsheng
Published: January 2023

Our understanding of cell-to-cell interactions has been significantly improved in the past years with the help of TIRFM in combination with an antigen presenting system supported by planar lipid bilayer (PLB) membranes. In TIRFM experiments, it is a challenge to uniformly present ligand molecules on the surface of PLB membranes. Here, we introduce a new and robust method of tethering IgG surrogate antigen ligands on the surface of PLB membranes containing nickel salt. In this method, we use a modified D domain from staphylococcal protein A molecule that is fused with an N-terminus polyhistidine tag (H12-D-domain) to tether IgG surrogate antigens on nickel-containing PLB membranes. We systemically assessed the specificity and capability of H12-D-domain construct to capture IgG molecules from different species through live cell and single molecule TIRFM imaging. We find that these IgG surrogate antigens show good lateral mobility and are uniformly distributed on PLB membranes. Not IgM molecules, nor Fab or F(ab’)2 fragments of IgG molecules can be tethered on PLB membranes by H12-D-domain construct. These tethered IgG surrogate antigens strongly induce the formation and accumulation of signaling active antigen receptor microclusters within the immunological synapse in B or T lymphocyte cells. Thus our method provides a new and robust way to tether IgG surrogate antigens or other molecule fused with IgG Fc portion on PLB membranes for TIRFM based molecule imaging experiments.