Cell Persistence Research Articles

Helicobacter pylori PldA modulates TNFR1-mediated p38 signaling pathways to regulate macrophage responses for its survival

ABSTRACT Helicobacter pylori, a dominant member of the gastric microbiota was associated with various gastrointestinal diseases and presents a significant challenge due to increasing antibiotic resistance. This study identifies H. pylori’s phospholipase A (PldA) as a critical factor in modulating host macrophage responses, facilitating H. pylori ‘s evasion of the immune system and persistence. PldA alters membrane lipids through reversible acylation and deacylation, affecting their structure and function. We found that PldA incorporates lysophosphatidylethanolamine into macrophage membranes, disrupting their bilayer structure and impairing TNFR1-mediated p38-MK2 signaling. This disruption results in reduced macrophage autophagy and elevated RIP1-dependent apoptosis, thereby enhancing H. pylori survival, a mechanism also observed in multidrug-resistant strains. Pharmacological inhibition of PldA significantly decreases H. pylori viability and increases macrophage survival. In vivo studies corroborate PldA’s essential role in H. pylori persistence and immune cell recruitment. Our findings position PldA as a pivotal element in H. pylori pathogenesis through TNFR1-mediated membrane modulation, offering a promising therapeutic target to counteract bacterial resistance.

Hypoimmune, Allogeneic CD22-Directed CAR T Cells That Evade Innate and Adaptive Immune Rejection for the Treatment of Large B Cell Lymphoma Patients That Are Relapsed/Refractory to CD19-Directed CAR T Cell Therapy

Despite the success of CD19CAR therapy against large B-cell lymphoma, up to 60% of CD19-directed CAR T cell treated patients relapse after, or are refractory to, treatment. Of the relapsed/refractory population a significant subset of patients, around 30%, have CD19-negative tumor and a short median survival prognosis - necessitating the implementation of rapid intervention strategies (Atilla, Transl Oncol 2022; Spiegel, Blood 2021). Sana is developing “off-the shelf” allogeneic CD22-directed CAR T cells that may enable rapid intervention and induce clinically meaningful activity for these CD19 CAR T cell relapsed/refractory patients. Here we describe methods to create hypoimmune (HIP), allogeneic CD22-directed CAR T cells using a clinically validated CD22CAR that: 1) support adaptive immune cell evasion via B2M and CIITA gene disruption, 2) promote persistence and innate immune cell evasion by overexpression of CD47, and 3) mitigate GvHD via TRAC gene disruption, (Hu, Nat Commun 2023). HIP, allogeneic CD22-directed CAR T cells (CD22 HIP CAR T cells) were derived from healthy donor CD8 + and CD4 + T cells that were gene-edited to disrupt the B2M, CIITA, and TRAC (TCR) genes and transduced to overexpress CD47 and express a second-generation, clinically validated CD22-directed CAR. Gene-edited Mock T cells and unedited, allogeneic CD47-CD22CAR T cells were generated as donor-matched controls. The CD22 HIP CAR T cells were evaluated for immune evasive activity using a previously described in vitro xCelligence cytotoxicity assay (Hu, Nat Commun 2023). The T cells were further evaluated in vitro for dose-dependent short-term and long-term cytolytic activity analysis via luciferase or IncuCyte assay, respectively. Correlate cytokine responses were performed by meso-scale discovery analysis and T cell expansion by IncuCyte classification. Systemic NALM and RAJI parental or CD19 antigen knockout tumor models (IV injection) were executed in a NSG or NSG-SGM3 mouse background. xCelligence analysis confirmed that CD22 HIP CAR T cells were protected from NK and macrophage recognition; whereas Mock control T cells that lack HLA expression succumbed to NK cell and macrophage killing within 5-hours of the 50-hour assay due to the absence of CD47 protection. Furthermore, CD22 HIP CAR T cells elicited cytotoxicity and robust cytokine responses (e.g. IFNγ, IL-2) in a dose-dependent manner comparable to allogeneic CD47-CD22CAR T cells when cultured with CD22 +/CD19 + or CD22 +/CD19 - NALM, RAJI, or K562 targets. The CD22 HIP CAR T cell and allogeneic CD47-CD22CAR T cell control produced low level IFNγ (<400pg/mL) and Granzyme B (<100pg/mL) when cultured alone or with CD22 - tumor targets. These basal levels of IFNγ and Granzyme B did not have long-term cytolytic effects on CD22 - tumor cells, nor was any significant level of IL-2 produced by CD22 HIP CAR T cells cultured alone, or in the presence of CD22-negative tumor targets. Indeed, CD22 HIP CAR T cell proliferation was dependent upon CD22 + tumor co-culture. NALM and RAJI CD19 knockout in vivo tumor models demonstrated that CD22 HIP CAR T cells significantly reduced flux (Day 21 AUC vs Mock: NALM and RAJI (p=0.0001)) and prolonged survival (Median vs Mock: p<0.004). Furthermore, CD22 HIP CAR T cells elicited comparable, dose-dependent antitumor activity relative to allogeneic CD47-CD22CAR T cell controls in a systemic NALM tumor model. Our data provide evidence that CD22 HIP CAR T cells elicit robust antitumor activity in an antigen and dose-dependent manner. Furthermore, these studies demonstrate that CD22 HIP CAR T cells are agnostic to CD19 expression and selectively elicit pharmacologic activity against, or in response to, CD22 + tumor. Results also demonstrate that the HIP approach to generate allogeneic CAR T cells that evade innate and adaptive immune rejection can be expanded as a platform to generate CAR T cells against target antigens alternative to CD19. Collectively, the data suggest that CD22 HIP CAR T cells display a combination of antigen-specific pharmacologic activity and immune evasion that support their progression into human clinical studies for the treatment of CD19 CAR T cell-refractory patients.

Microbial Growth under Limiting Conditions-Future Perspectives.

Microorganisms rule the functioning of our planet and each one of the individual macroscopic living creature. Nevertheless, microbial activity and growth status have always been challenging tasks to determine both in situ and in vivo. Microbial activity is generally related to growth, and the growth rate is a result of the availability of nutrients under adequate or adverse conditions faced by microbial cells in a changing environment. Most studies on microorganisms have been carried out under optimum or near-optimum growth conditions, but scarce information is available about microorganisms at slow-growing states (i.e., near-zero growth and maintenance metabolism). This study aims to better understand microorganisms under growth-limiting conditions. This is expected to provide new perspectives on the functions and relevance of the microbial world. This is because (i) microorganisms in nature frequently face conditions of severe growth limitation, (ii) microorganisms activate singular pathways (mostly genes remaining to be functionally annotated), resulting in a broad range of secondary metabolites, and (iii) the response of microorganisms to slow-growth conditions remains to be understood, including persistence strategies, gene expression, and cell differentiation both within clonal populations and due to the complexity of the environment.

3D-organoid culture supports differentiation of human CAR+ iPSCs into highly functional CAR Tcells.

Unlimited generation of chimeric antigen receptor (CAR) Tcells from human-induced pluripotent stem cells (iPSCs) is an attractive approach for "off-the-shelf" CAR Tcell immunotherapy. Approaches to efficiently differentiate iPSCs into canonical αβ Tcell lineages, while maintaining CAR expression and functionality, however, have been challenging. We report that iPSCs reprogramed from CD62L+ naive and memory Tcells followed by CD19-CAR engineering and 3D-organoid system differentiation confers products with conventional CD8αβ-positive CAR Tcell characteristics. Expanded iPSC CD19-CAR Tcells showed comparable antigen-specific activation, degranulation, cytotoxicity, and cytokine secretion compared with conventional CD19-CAR Tcells and maintained homogeneous expression of the TCR derived from the initial clone. iPSC CD19-CAR Tcells also mediated potent antitumor activity invivo, prolonging survival of mice with CD19+ human tumor xenografts. Our study establishes feasible methodologies to generate highly functional CAR Tcells from iPSCs to support the development of "off-the-shelf" manufacturing strategies.

Exotoxin S secreted by internalized Pseudomonas aeruginosa delays lytic host cell death.

The Pseudomonas aeruginosa toxin ExoS, secreted by the type III secretion system (T3SS), supports intracellular persistence via its ADP-ribosyltransferase (ADPr) activity. For epithelial cells, this involves inhibiting vacuole acidification, promoting vacuolar escape, countering autophagy, and niche construction in the cytoplasm and within plasma membrane blebs. Paradoxically, ExoS and other P. aeruginosa T3SS effectors can also have antiphagocytic and cytotoxic activities. Here, we sought to reconcile these apparently contradictory activities of ExoS by studying the relationships between intracellular persistence and host epithelial cell death. Methods involved quantitative imaging and the use of antibiotics that vary in host cell membrane permeability to selectively kill intracellular and extracellular populations after invasion. Results showed that intracellular P. aeruginosa mutants lacking T3SS effector toxins could kill (permeabilize) cells when extracellular bacteria were eliminated. Surprisingly, wild-type strain PAO1 (encoding ExoS, ExoT and ExoY) caused cell death more slowly, the time extended from 5.2 to 9.5 h for corneal epithelial cells and from 10.2 to 13.0 h for HeLa cells. Use of specific mutants/complementation and controls for initial invasion showed that ExoS ADPr activity delayed cell death. Triggering T3SS expression only after bacteria invaded cells using rhamnose-induction in T3SS mutants rescued the ExoS-dependent intracellular phenotype, showing that injected effectors from extracellular bacteria were not required. The ADPr activity of ExoS was further found to support internalization by countering the antiphagocytic activity of both the ExoS and ExoT RhoGAP domains. Together, these results show two additional roles for ExoS ADPr activity in supporting the intracellular lifestyle of P. aeruginosa; suppression of host cell death to preserve a replicative niche and inhibition of T3SS effector antiphagocytic activities to allow invasion. These findings add to the growing body of evidence that ExoS-encoding (invasive) P. aeruginosa strains can be facultative intracellular pathogens, and that intracellularly secreted T3SS effectors contribute to pathogenesis.

DOP19 Myeloid and lymphoid cell abnormalities persist in the intestinal mucosa of patients who recover from COVID-19 infection

BackgroundAlthough respiratory failure is the hallmark of severe disease, it is increasingly clear that Coronavirus Disease 2019 (COVID-19) is a multi-system disorder. The presence of gastrointestinal (Gl) involvement by Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has been suggested by epidemiological, clinical, non-human primate, in-vitro (enteroid) and ex-vivo (human biopsy) studies. Having recently documented persistence of SAR-CoV-2 within the intestinal epithelium 7 months after infection, here we aimed to study mucosal immune cell abnormalities in individuals with prior history of COVID-19.MethodsIndividuals with previous COVID-19 diagnosis (by either RT–PCR or seroconversion) and controls (without RT-PCR or serological evidence of prior COVID-19 infection) undergoing endoscopic evaluation were recruited into the study (Table 1,2). Colonic and small intestinal (duodenal and ileal) biopsies were analyzed by multiparameter flow cytometry for mucosal immune cell populations including myeloid cells (classical and non-classical monocytes, dendritic cell subsets), T cells (subsets and activation state), B cells (including plasma cells). Persistence of viral antigens was determined by immunofluorescence microscopy (n=30) using a previously published anti-nucleocapsid (NP) antibody.ResultsThirty subjects with a previous history of COVID-19 (post-COVID), median of 4 months from diagnosis (range 1–10 months), were recruited and compared with 40 normal volunteer (NV) controls. Relative to controls, post-COVID subjects displayed higher frequencies of classical (CD14+) monocytes in both, the colon and the small bowel, while significantly higher frequencies of conventional dendritic cells (cDC) 1 (lin-HLA-DRhiCD14-CD11c+CD141+) and cDC2 (lin-HLA-DRhiCD14--CD11c+CD1c+) were noted in the colon only. Among T cell subsets, CD8+ tissue resident memory T cells (CD8+CD69+CD103+) were significantly increased in colon of post-COVID subjects compared to NV. Among B cell subsets, plasma cells (CD3-CD27+CD38hi) trended higher (p=0.06), while mucosal B cells (CD3-CD19+) were significantly lower in the terminal ileum of post-COVID subjects compared to NV. Finally, with IF, we detected SARS-CoV-2 NP in 10 out of 30 (33%) of post-COVID subjects (Figure 1). There were no significant correlations of these cell populations with either time after the infection or IF positivity. ConclusionInnate and adaptive immune cell abnormalities persist in the intestinal mucosa of post-COVID subjects for up to 10 months and may reflect viral persistence or immune cell dysregulation in the intestines. These findings have major implications for understanding the pathogenesis of long term sequela of COVID-19, including long-haul COVID.

Engineered Hydroxyapatite Nanoadjuvants with Controlled Shape and Aspect Ratios Reveal Their Immunomodulatory Potentials.

Hydroxyapatite (HAP) has been formulated as adjuvants in vaccines for human use. However, the optimal properties required for HAP nanoparticles to elicit adjuvanticity and the underlying immunopotentiation mechanisms have not been fully elucidated. Herein, a library of HAP nanorods and nanospheres was synthesized to explore the effect of the particle shape and aspect ratio on the immune responses in vitro and adjuvanticity in vivo. It was demonstrated that long aspect ratio HAP nanorods induced a higher degree of cell membrane depolarization and subsequent uptake, and the internalized particles elicited cathepsin B release and mitochondrial reactive oxygen species generation, which further led to pro-inflammatory responses. Furthermore, the physicochemical property-dependent immunostimulation capacities were correlated with their humoral responses in a murine hepatitis B surface antigen immunization model, with long aspect ratio HAP nanorods inducing higher antigen-specific antibody productions. Importantly, HAP nanorods significantly up-regulated the IFN-γ secretion and CD107α expression on CD8+ T cells in immunized mice. Further mechanistic studies demonstrated that HAP nanorods with defined properties exerted immunomodulatory effects by enhanced antigen persistence and immune cell recruitments. Our study provides a rational design strategy for engineered nanomaterial-based vaccine adjuvants.

Genotypic and Phenotypic Characterization of Incompatibility Group FIB Positive Salmonella enterica Serovar Typhimurium Isolates from Food Animal Sources.

Salmonella enterica is one of the most common bacterial foodborne pathogens in the United States, causing illnesses that range from self-limiting gastroenteritis to more severe, life threatening invasive disease. Many Salmonella strains contain plasmids that carry virulence, antimicrobial resistance, and/or transfer genes which allow them to adapt to diverse environments, and these can include incompatibility group (Inc) FIB plasmids. This study was undertaken to evaluate the genomic and phenotypic characteristics of IncFIB-positive Salmonella enterica serovar Typhimurium isolates from food animal sources, to identify their plasmid content, assess antimicrobial resistance and virulence properties, and compare their genotypic isolates with more recently isolated S. Typhimurium isolates from food animal sources. Methods: We identified 71 S. Typhimurium isolates that carried IncFIB plasmids. These isolates were subjected to whole genome sequencing and evaluated for bacteriocin production, antimicrobial susceptibility, the ability to transfer resistance plasmids, and a subset was evaluated for their ability to invade and persist in intestinal human epithelial cells. Results: Approximately 30% of isolates (n = 21) displayed bacteriocin inhibition of Escherichia coli strain J53. Bioinformatic analyses using PlasmidFinder software confirmed that all isolates contained IncFIB plasmids along with multiple other plasmid replicon types. Comparative analyses showed that all strains carried multiple antimicrobial resistance genes and virulence factors including iron acquisition genes, such as iucABCD (75%), iutA (94%), sitABCD (76%) and sitAB (100%). In 17 cases (71%), IncFIB plasmids, along with other plasmid replicon types, were able to conjugally transfer antimicrobial resistance and virulence genes to the susceptible recipient strain. For ten strains, persistence cell counts (27%) were noted to be significantly higher than invasion bacterial cell counts. When the genome sequences of the study isolates collected from 1998–2003 were compared to those published from subsequent years (2005–2018), overlapping genotypes were found, indicating the perseverance of IncFIB positive strains in food animal populations. This study confirms that IncFIB plasmids can play a potential role in disseminating antimicrobial resistance and virulence genes amongst bacteria from several food animal species.

Compaction Dynamics during Progenitor Cell Self-Assembly Reveal Granular Mechanics

Summary We study the self-assembly dynamics of human progenitor cells in agarose microwells that are used for production of chondrogenic organoids. Using image analysis on time-lapse microscopy, we estimate the aggregate area in function of time for a large number of aggregates. In control conditions, the aggregate radius follows an exponential relaxation that is consistent with the dewetting dynamics of a liquid film. Upon introducing Y-27632 Rho kinase inhibitor, the compatibility with the liquid model is lost, and slowed-down relaxation dynamics are observed. We demonstrate that these aggregates behave as granular piles undergoing compaction, with density relaxation that follows a stretched exponential. Using simulations with an individual cell-based model, we construct a phase diagram of cell aggregates that suggests that the aggregate in presence of Rho kinase inhibitor approaches glass transition.

Abstract 3208: Using T cell engineering plus triple checkpoint blockade to enhance the efficacy of adoptive immunotherapy in ovarian cancer

Abstract Over 20,000 women are diagnosed with ovarian cancer in the United States annually, and over half will die within 5 years. Outcomes have changed little in the last 20 years, highlighting the need for therapy innovation. One promising new strategy employs immune T cells engineered to target proteins uniquely overexpressed in tumors; such T cell immunotherapies have the potential to control tumor growth without toxicity to healthy tissues. In considering candidate immunotherapy targets, we focused on mesothelin (MSLN), which contributes to invasive progression and malignancy in ovarian cancer but has limited expression in healthy cells. We showed that T cells engineered to express a human or mouse MSLN-specific high-affinity T cell receptor (TCRMSLN) can kill human patient-derived ovarian cancer cell lines or the murine ID8 cell line, respectively. In a disseminated ID8 tumor model, adoptively transferred TCRMSLN T cells preferentially accumulated within established tumors, delayed ovarian tumor growth, and significantly prolonged mouse survival. However, our data also revealed that the ovarian tumor microenvironment (TME) limits engineered T cell persistence and cancer cell killing. To identify immunosuppressive features active in both the human and murine ovarian TME, we performed gene expression analyses. Deep transcriptome profiling confirmed similar gene expression signatures in human cancers and in the preclinical ID8 model. Among these, RNA sequencing detected PD-L1, Galectin-9 and Galectin-3, ligands for CD8 T cell-expressed PD-1, Tim-3 and Lag-3 ‘checkpoint’ receptors, respectively. We also measured PD-L1, Galectin-9 and Galectin-3 expression in human and mouse ovarian cancers by flow cytometry and immunohistochemistry, and multiplex immunohistochemistry of human ovarian tumors confirmed the presence of endogenous CD8 T cells expressing one, two or all three inhibitory receptors. Moreover, flow cytometry revealed that TCRMSLN-transduced T cells increase expression of the inhibitory receptors in ID8 tumors relative to cells in the spleen as early as three weeks after transfer, in association with decreased production of anti-tumor cytokines. Based on our results, we hypothesized that we could overcome engineered T cell suppression via inhibitory receptor ligation. We treated tumor-bearing mice with TCRMSLN T cells plus anti-PD-1, anti-Tim-3 and/or anti-Lag-3 checkpoint-blocking antibodies, targeting up to three inhibitory receptors simultaneously. Triple checkpoint blockade dramatically increased anti-tumor cytokine production by intratumoral TCRMSLN T cells. As many solid tumors both overexpress MSLN as well as PD-1, Tim-3 and Lag-3, the use of multi-checkpoint blockade with engineered T cells has real potential to also enhance the efficacy of engineered adoptive T cell therapy against other malignancies. Citation Format: Kristin G. Anderson, Madison G. Burnett, Valentin Voillet, Edison Y. Chiu, Breanna M. Bates, Nicolas M. Garcia, Raphael Gottado, Philip D. Greenberg. Using T cell engineering plus triple checkpoint blockade to enhance the efficacy of adoptive immunotherapy in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3208.

Chimeric antigen receptor T cell persistence and memory cell formation.

It is now becoming clear that less differentiated naive and memory T cells are superior to effector T cells in the transfer of immunity for adoptive cell therapy. This review will outline the challenges faced by chimeric antigen receptor (CAR) Tcell therapy in the generation of persistence and memory for CAR T cells, and summarize recent strategies to improve CAR Tcell persistence, with a focus on memory cell formation. The relevance of enhancing persistence in more differentiated effector T cells is also covered, because genetic and pharmacological interventions may prolong effector Tcell activity and lifespan, thereby improving anti-cancer activity. In particular, it may be possible to enforce epigenetic changes in differentiated T cells to enhance memory CAR Tcell formation. Optimizing the generation of self-renewing Tcell populations (e.g. memory cells), while maintaining differentiated effector T cells through epigenome modification, will help overcome barriers to Tcell expansion and survival, thereby improving clinical outcomes in CAR Tcell therapy.

Mechanisms of resistance to CAR T cell therapy.

The successes with chimeric antigen receptor (CAR) T cell therapy in early clinical trials involving patients with pre-B cell acute lymphoblastic leukaemia (ALL) or B cell lymphomas have revolutionized anticancer therapy, providing a potentially curative option for patients who are refractory to standard treatments. These trials resulted in rapid FDA approvals of anti-CD19 CAR T cell products for both ALL and certain types of B cell lymphoma - the first approved gene therapies in the USA. However, growing experience with these agents has revealed that remissions will be brief in a substantial number of patients owing to poor CAR T cell persistence and/or cancer cell resistance resulting from antigen loss or modulation. Furthermore, the initial experience with CAR T cells has highlighted challenges associated with manufacturing a patient-specific therapy. Understanding the limitations of CAR T cell therapy will be critical to realizing the full potential of this novel treatment approach. Herein, we discuss the factors that can preclude durable remissions following CAR T cell therapy, with a primary focus on the resistance mechanisms that underlie disease relapse. We also provide an overview of potential strategies to overcome these obstacles in an effort to more effectively incorporate this unique therapeutic strategy into standard treatment paradigms.

TMOD-01. THE ROLE OF PERSISTENCE, PROLIFERATION, AND TUMOR CELL KILLING EFFICIENCY IN DETERMINING RESPONSE TO CAR T-CELL THERAPY IN GLIOBLASTOMA: A MATHEMATICAL MODEL AND ANALYSIS

Chimeric antigen receptor (CAR) T-cell therapy is a promising emerging area of immunotherapy for the treatment of recurrent glioblastoma. In this therapy, T-cells are genetically modified to target tumor-specific antigens. Efficacy of CAR T-cell therapy depends on several factors, including CAR T-cell proliferation, persistence, and tumor cell killing capacity. Here we use a mathematical model to investigate the role of these three factors in determining response of recurrent GBM to this novel immunotherapy. Additionally, the impact of dosing and scheduling of CAR T-cell infusions on the therapeutic response is explored. We model the interaction between the two cell populations (cancer cells and CAR T-cells) using an ordinary differential equation based formalism. The growth and death of cancer cells are simulated as rates of proliferation and interaction between cancer cells and CAR T-cells, respectively. Biological data was used to parameterize the model. Analysis of the dynamics of interaction between cancer cells and CAR T-cells was performed to determine the maximum efficacy of a single and multiple doses of CAR T-cells. Our mathematical model and analysis shows that a critical parameter for the success of CAR T-cell therapy is the ratio of cancer cell proliferation to the killing capacity of the CAR T-cells. We quantify the dose level of CAR T-cells required to eliminate the cancer cell population. Furthermore, we use the mathematical model to predict the time to progression for specific dose levels, which may help in optimizing and scheduling multiple doses of CAR T-cell monotherapy. We compare the mathematical results with patient data from an ongoing dose-escalation study (NCT02208362) using central memory derived IL13Rα2-targeted CAR T-cell therapy for recurrent glioblastoma.

Integrated analysis of HPV-mediated immune alterations in cervical cancer

ObjectiveHuman papillomavirus (HPV) infection is the primary cause of cervical cancer. HPV-mediated immune alterations are known to play crucial roles in determining viral persistence and host cell transformation. We sought to thoroughly understand HPV-directed immune alterations in cervical cancer by exploring publically available datasets. Methods130 HPV positive and 7 HPV negative cervical cancer cases from The Cancer Genome Atlas were compared for differences in gene expression levels and functional enrichment. Analyses for copy number variation (CNV) and genetic mutation were conducted for differentially expressed immune genes. Kaplan-Meier analysis was performed to assess survival and relapse differences across cases with or without alterations of the identified immune signature genes. ResultsGenes up-regulated in HPV positive cervical cancer were enriched for various gene ontology terms of immune processes (P=1.05E-14~1.00E-05). Integrated analysis of the differentially expressed immune genes identified 9 genes that displayed either CNV, genetic mutation and/or gene expression changes in at least 10% of the cases of HPV positive cervical cancer. Genomic amplification may cause elevated levels of these genes in some HPV positive cases. Finally, patients with alterations in at least one of the nine signature genes overall had earlier relapse compared to those without any alterations. The altered expression of either TFRC or MMP13 may indicate poor survival for a subset of cervical cancer patients (P=1.07E-07). ConclusionsWe identified a novel immune gene signature for HPV positive cervical cancer that is potentially associated with early relapse of cervical cancer.

Discrete Modeling of Amoeboid Locomotion and Chemotaxis in Dictyostelium discoideum by Tracking Pseudopodium Growth Direction

Dictyostelium discoideum amoeba is a well-established model organism for studying the crawling locomotion of eukaryotic cells. These amoebae extend pseudopodium - a temporary actin-based protrusion of their body membrane to probe the medium and crawl through it. Experiments show highly-ordered patterns in the growth direction of these pseudopodia, which results in persistence cell motility. Here, we propose a discrete model for studying and investigating the cell locomotion based on the experimental evidences. According to our model, Dictyostelium selects its pseudopodium growth direction based on a second-order Markov chain process, in the absence of external cues. Consequently, compared to a random walk process, our model indicates stronger growth in the mean-square displacement of cells, which is consistent with empirical findings. In the presence of external chemical stimulants, cells tend to align with the gradient of chemoattractant molecules. To quantify this tendency, we define a coupling coefficient between the pseudopodium extension direction and the gradient of an external stimulant, which depends on the local stimulant concentration and its gradient. Additionally, we generalize the model to weak-coupling regime by utilizing perturbation methods.

Editorial to the Research Topic "Comparative studies between HTLV-1 and HTLV-2 function and pathobiology".

Editorial to the Research Topic "Comparative studies between HTLV-1 and HTLV-2 function and pathobiology".

1037-LBP: Lineage marker “Negative” Fraction resolves discrepancy in lineage specific chimerism

Aim Donor Chimerism (DC) analysis documents engraftment, disease persistence and/or recurrence post allogeneic hematopoietic stem cell transplantation (HSCT). Typically, unfractionated analysis (UA) is performed but lineage-specific chimerism analysis (FC) can document specific cell engraftment or early relapse. However, loss or alteration of cell surface marker expression may hamper selection of relapsed cells and results inaccurately show only donor genotype. We present two Acute Myelogenous Leukemia (AML) patients with discordant results between FC and UA where analysis of Negative Fraction (NF) furnished evidence of relapse. Purpose Demonstrate utility of NF analysis to resolve discrepant UA/FC results. Methods DC was evaluated using commercially available STR reagents (Life Tech. and Promega). Sequential separation of B, T and myeloid cells was performed on post HSCT blood (RoboSep, StemCell, Inc.). Cells remaining after selection were saved, i.e. Negative Fraction (NF). Chimerism analysis was performed on UA and cell fraction DNA. NF was analyzed if discrepant results between UA and FC were found. Results HSCT patients were tested for DC at 30 days post txp and for cause. Pt#1, whose 30 day analysis was missed, was tested 5 months post txp. FC showed no B cells, > 95% DC in T and myeloid fractions, but 11% DC in UA. NF was predominantly recipient genotype. For Pt#2, 90 day analysis showed 19.5 % DC in UA, > 92% in T and myeloid fractions with negligible B cells. NF showed 9% DC. In both patients, relapsed cells escaped the fractionation process. Both patients were diagnosed with relapse. Conclusions FC is useful in detecting relapse post HSCT but recurrent tumor cells may not express separation markers and results will be inaccurate. Concurrent UA analysis is recommended. NF testing may resolve discrepancies between UA and FC.

Intraperitoneal delivery of human natural killer cells for treatment of ovarian cancer in a mouse xenograft model

Background aimsThere is an urgent need for novel therapeutic strategies for relapsed ovarian cancer. Dramatic clinical anti-tumor effects have been observed with interleukin (IL)-2 activated natural killer (NK) cells; however, intravenous delivery of NK cells in patients with ovarian cancer has not been successful in ameliorating disease. We investigated in vivo engraftment of intraperitoneally (IP) delivered NK cells in an ovarian cancer xenograft model to determine if delivery mode can affect tumor cell killing and circumvent lack of NK cell expansion. MethodsAn ovarian cancer xenograft mouse model was established to evaluate efficacy of IP-delivered NK cells. Tumor burden was monitored by bioluminescent imaging of luciferase-expressing ovarian cancer cells. NK cell persistence, tumor burden and NK cell trafficking were evaluated. Transplanted NK cells were evaluated by flow cytometry and cytotoxicity assays. ResultsIP delivery of human NK cells plus cytokines led to high levels of circulating NK and was effective in clearing intraperitoneal ovarian cancer burden in xenografted mice. NK cells remained within the peritoneal cavity 54 days after injection and had markers of maturation. Additionally, surviving NK cells were able to kill ovarian cancer cells at a rate similar to pre-infusion levels, supporting that in vivo functionality of human NK cells can be maintained after IP infusion. ConclusionsIP delivery of NK cells leads to stable engraftment and antitumor response in an ovarian cancer xenograft model. These data support further pre-clinical and clinical evaluation of IP delivery of allogeneic NK cells in ovarian cancer.

FRI0259 Ectopic lymphoid structures support epstein-barr virus persistence and autoreactive plasma cell infection in sjogren’s syndrome salivary glands

BackgroundThe ubiquitous □-herpesvirus Epstein-Barr virus (EBV) infects B cells and modifies their differentiation programme leading to B cell activation and immortalization. Due to these unique properties, EBV has been suggested...

PTP1B promotes focal complex maturation, lamellar persistence and directional migration

Previous findings established that ER-bound PTP1B targets peripheral cell-matrix adhesions and positively regulates cell adhesion to fibronectin. Here we show that PTP1B enhances focal complex lifetime at the lamellipodium base, delaying their turnover and facilitating α-actinin incorporation. We demonstrate the presence of catalytic PTP1BD181A-α-actinin complexes at focal complexes. Kymograph analysis revealed that PTP1B contributes to lamellar protrusion persistence and directional cell migration. Pull-down and FRET analysis also showed that PTP1B is required for efficient integrin-dependent downregulation of RhoA and upregulation of Rac1 during spreading. A substrate trap strategy revealed that FAK/Src recruitment and Src activity are essential for the generation of PTP1B substrates in adhesions. PTP1B targets the negative regulatory site of Src (phosphotyrosine 529), paxillin and p130Cas at peripheral cell-matrix adhesions. We postulate that PTP1B modulates more than one pathway required for focal complex maturation and membrane protrusion, including α-actinin-mediated cytoskeletal anchorage, integrin-dependent activation of the FAK/Src signaling pathway, and RhoA and Rac1 GTPase activity. By doing so, PTP1B contributes to coordinated adhesion turnover, lamellar stability and directional cell migration.