TM Cells Research Articles

The Organization of the Second Optic Chiasm of the Drosophila Optic Lobe.

Visual pathways from the compound eye of an insect relay to four neuropils, successively the lamina, medulla, lobula, and lobula plate in the underlying optic lobe. Among these neuropils, the medulla, lobula, and lobula plate are interconnected by the complex second optic chiasm, through which the anteroposterior axis undergoes an inversion between the medulla and lobula. Given their complex structure, the projection patterns through the second optic chiasm have so far lacked critical analysis. By densely reconstructing axon trajectories using a volumetric scanning electron microscopy (SEM) technique, we reveal the three-dimensional structure of the second optic chiasm of Drosophila melanogaster, which comprises interleaving bundles and sheets of axons insulated from each other by glial sheaths. These axon bundles invert their horizontal sequence in passing between the medulla and lobula. Axons connecting the medulla and lobula plate are also bundled together with them but do not decussate the sequence of their horizontal positions. They interleave with sheets of projection neuron axons between the lobula and lobula plate, which also lack decussations. We estimate that approximately 19,500 cells per hemisphere, about two thirds of the optic lobe neurons, contribute to the second chiasm, most being Tm cells, with an estimated additional 2,780 T4 and T5 cells each. The chiasm mostly comprises axons and cell body fibers, but also a few synaptic elements. Based on our anatomical findings, we propose that a chiasmal structure between the neuropils is potentially advantageous for processing complex visual information in parallel. The EM reconstruction shows not only the structure of the chiasm in the adult brain, the previously unreported main topic of our study, but also suggest that the projection patterns of the neurons comprising the chiasm may be determined by the proliferation centers from which the neurons develop. Such a complex wiring pattern could, we suggest, only have arisen in several evolutionary steps.

Abstract CT160: A Phase I, open-label, safety, pharmacokinetic, and preliminary efficacy study of STRO-002, an anti-folate receptor alpha (FolRα) antibody drug conjugate (ADC), in patients with advanced epithelial ovarian cancer (including fallopian tube or primary peritoneal cancers) and endometrial cancers

Abstract Background: Folate receptor alpha (FolRα) is overexpressed in 80% of epithelial ovarian cancer (EOC) and endometrial adenocarcinoma (EC), with minimal expression on normal tissues and is thus a relevant target for cancer therapy using antibody drug conjugates (ADCs). STRO-002 is a novel, site-specific FolRα-targeting ADC that was generated using Sutro’s XpressCF+™ cell free protein synthesis platform. STRO-002 has highly specific cytotoxic activity on cells expressing FolRα. STRO-002 demonstrated reproducible and significant therapeutic efficacy in multiple preclinical models in vivo. STRO-002 activity was evaluated in EC patient-derived xenograft (PDX) models with a range of FolRα expression. Statistically significant efficacy was observed in 60% of the FolRα-positive PDX models tested, with response appearing to positively correlate with FolRα expression levels. Toxicology studies in cynomolgus monkeys demonstrate dose-dependent and reversible neutropenia when STRO-002 is administered at up to 9 mg/kg, with no evidence of ocular toxicity (Solis et al. AACR 2019). Methods: This study is a first-in-human Phase 1, open-label, multicenter, dose escalation (Part 1) study with dose expansion (Part 2) to identify the maximum tolerated dose (MTD), recommended phase 2 doses (RP2D) and to evaluate the safety, tolerability, and preliminary anti-tumor activity of STRO-002 in adults with ovarian cancer and endometrial cancer who are refractory to, or intolerant of, therapies known to provide clinical benefit. STRO-002 is given to all patients on study via intravenous infusion on Day 1of each 21-day cycle until disease progression. Dose limiting toxicities will be assessed in the first cycle (Days 1-21) of dose escalation. Part 1 will enroll about 30 ovarian cancer patients to determine the MTD and RP2D for expansion while Part 2 will enroll 2 cohorts based on disease subtypes (ovarian or endometrial). Efficacy will be evaluated per RECIST 1.1 criteria. Key inclusion criteria include relapsed and or progressive disease, adequate bone marrow and renal function, and ability to comply with treatment, testing and pharmacokinetic (PK) schedules. Key exclusion criteria include clear cell, mucinous or sarcomatous ovarian carcinoma, prior treatment with a FolRα-targeting ADC, need for ongoing immunotherapy including systemic corticosteroids and a history of CNS involvement. Samples will be collected to assess FolRα expression levels, PK and immunogenicity. No formal statistical hypothesis testing will be conducted in this study. This study is currently open for enrollment in the US with the ClinicalTrials.gov Identifier: NCT03748186.: Citation Format: Robert Wendel Naumann, Denise S. Uyar, Russell J. Schilder, Michael A. Palumbo, Venita DeAlmeida, Shannon L. Matheny, Arturo Molina. A Phase I, open-label, safety, pharmacokinetic, and preliminary efficacy study of STRO-002, an anti-folate receptor alpha (FolRα) antibody drug conjugate (ADC), in patients with advanced epithelial ovarian cancer (including fallopian tube or primary peritoneal cancers) and endometrial cancers [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 CT160.

POSSIBLE PREVENTION OF REACTIVE OXYGEN SPECIES INDUCED HUMAN TRABECULAR MESHWORK CELL DAMAGE BY RESVERATROL AND ASCORBIC ACID.

Objectives: To analyze the antioxidant activity of Resveratrol and Ascorbic Acid against hydrogen peroxide (an oxidant) mediated cell injury of human trabecular meshwork cells. Study Design: Experimental study. Setting: Molecular Biology Laboratory at Medical Research Centre, Liaquat University of Medical & Health Sciences, Jamshoro. Period: Six months. Materials and Methods: Human Trabecular Meshwork cells were purchased from ScienCell Research Laboratories, USA. TM cell metabolism, TM cell viability and Reactive oxygen species were detected by standard methods in co- and pre- treated TM cells. Results: A significant reduction in TM cell metabolism was observed approximating 61% at 1.0 mM H2O2 compared to Ascorbate – 99% and Resveratrol 99% (p=0.0001). Resveratrol was more effective than Ascorbate even at 4.0 mM H2O2, the TM cell activity was noted 76%. Compared to H2O2- treated TM cells, resveratrol improved mitochondrial function upto 4.0 mM H2O2 (76%). Compared to co-treatment, the pretreatment shows similar results except at 4.0 mM H2O2. At 4.0 mM H2O2 the pre-treat TM cell metabolic activity was found as 11%, 31% and 47% compared to co-treat as 9%, 31% and 76% in controls, ascorbate and resveratrol groups respectively (p<0.05). Resveratrol shows significant decrease in viability was seen in controls compared to Ascorbate and Resveratrol groups. Cell viability showed statistically significant differences at 2.0 and 4.0 mM H2O2 compared to controls (P=0.0001). For reactive oxygen species (ROS), cells were incubated and with Ascorbate and Resveratrol for 24 hours and TM cells were treated with 0.0mM, 0.5 mM, 1.0 mM, 2.0mM and 4.0mM H2O2. Significant decrease in ROS was noted by Resveratrol compared to Ascorbate. Conclusions: Resveratrol and Ascorbate may prove useful in preventing and delaying the glaucoma, and timely institution of these anti – oxidants may help maintain trabecular meshwork functions and prevent visual loss.

Zika Virus Infects Trabecular Meshwork and Causes Trabeculitis and Glaucomatous Pathology in Mouse Eyes.

Zika virus (ZIKV) infection during pregnancy leads to devastating fetal outcomes, including neurological (microcephaly) and ocular pathologies such as retinal lesions, optic nerve abnormalities, chorioretinal atrophy, and congenital glaucoma. Only clinical case reports have linked ZIKV infection to causing glaucoma, a major blinding eye disease. In the present study, we have investigated the role of ZIKV in glaucoma pathophysiology using in vitro and in vivo experimental models. We showed that human primary trabecular meshwork (Pr. TM) cells, as well as a human GTM3 cell line, were permissive to ZIKV infection. ZIKV induced the transcription of various genes expressing pattern recognition receptors (TLR2, TLR3, and RIG-I), cytokines/chemokines (TNF-α, IL-1β, CCL5, and CXCL10), interferons (IFN-α2, IFN-β1, and IFN-γ), and interferon-stimulated genes (ISG15 and OAS2) in Pr. TM cells. ZIKV infection in IFNAR1-/- and wild-type (WT) mouse eyes resulted in increased intraocular pressure (IOP) and the development of chorioretinal atrophy. Anterior chamber (AC) inoculation of ZIKV caused infectivity in iridocorneal angle and TM, leading to the death of TM cells in the mouse eyes. Moreover, anterior segment tissue of infected eyes exhibited increased expression of inflammatory mediators and interferons. Furthermore, ZIKV infection in IFNAR1-/- mice resulted in retinal ganglion cell (RGC) death and loss, coinciding with optic nerve infectivity and disruption of anterograde axonal transport. Because of similarity in glaucomatous pathologies in our study and other experimental glaucoma models, ZIKV infection can be used to study infectious triggers of glaucoma, currently an understudied area of investigation.IMPORTANCE Ocular complications due to ZIKV infection remains a major public health concern because of their ability to cause visual impairment or blindness. Most of the previous studies have shown ZIKV-induced ocular pathology in the posterior segment (i.e., retina) of the eye. However, some recent clinical reports from affected countries highlighted the importance of ZIKV in affecting the anterior segment of the eye and causing congenital glaucoma. Because glaucoma is the second leading cause of blindness worldwide, it is imperative to study ZIKV infection in causing glaucoma to identify potential targets for therapeutic intervention. In this study, we discovered that ZIKV permissively infects human TM cells and evokes inflammatory responses causing trabeculitis. Using a mouse model, we demonstrated that ZIKV infection resulted in higher IOP, increased RGC loss, and optic nerve abnormalities, the classical hallmarks of glaucoma. Collectively, our study provides new insights into ocular ZIKV infection resulting in glaucomatous pathology.

Idiopathic inflammatory myopathy patients with different autoantibody exhibit unique peripheral blood T cell subsets

Abstract Idiopathic inflammatory myopathy (IIM), a heterogeneous group of systemic autoimmune diseases, is characterized clinically by weakness of skeletal muscle. Patients with IIM often show a myositis-specific autoantibody (MSA) such as anti-melanoma differentiation-associated gene-5 (MDA5 group) or anti-aminoacyl-tRNA synthetase (ARS group, including Jo-1, EJ, PL-12, PL-7, OJ, and KS), indicating that the adaptive immune system may alter in IIM patients. T cells (CD3+) play important roles in regulation of the adaptive immune system and usually subdivide into naïve cells (TN) and memory T cells (TM). Based on the surface expression of different cell markers, TM cells can be divided into central memory T (TCM, CD45RA−CD62L+) and effector memory T (TEM, CD45RA−CD62L−) cells. After antigenic stimulation, a subset of TEM cells re-expresses CD45RA, called TTEMRA. Here we examined peripheral blood T cell subsets in MDA5 group (n=22), ARS group (n=34) and healthy control individuals (HC group, n=30) using flow cytometry analysis. We found that TTEMRA population was increased in MDA5 group. TN population was reduced and TTEMRA population was enhanced in ARS group. Th17 and Th1 cells were more abundant in MDA5 and in ARS group, respectively. Treg cells was increased in both of MDA5 and ARS group. In addition, in MDA5 group, death patients (n=5) had fewer Treg cells compared with alive patients (n=17), suggesting reduced Treg cells may be associated with mortality. Therefore, peripheral blood T cell subsets may be correlated with specific clinical manifestations for IIM patients with distinct MSA.

A nanoparticle-based approach targeting ion channels for the treatment of Lupus nephritis

Abstract Lupus nephritis (LN) occurs in up to 60% of patients with systemic lupus erythematosus (SLE) and it remains the predominant cause of morbidity and mortality. Current available therapies for SLE have serious side effects and limited efficacy. Our research aims to develop a novel therapy for SLE with improved efficacy and minimal side effects. SLE is in large part driven by hyperactive T cells, which present with multiple defects including increased INFγ, chemokine receptors and CD40L that exacerbate tissue infiltration, inflammation and B cell stimulation. Kv1.3 channels regulate the Ca2+ signaling necessary for these effector functions. We have developed lipid nanoparticles that selectively target memory T (TM) cells and reduce their hyper-functionality by downregulating Kv1.3 via siRNAs (Kv1.3-NPs). Our in-vitro data showed that Kv1.3-NPs suppressed Ca2+ signaling, CD40L expression and IFNγ release selectively in TM cells. To demonstrate therapeutic efficacy of the Kv1.3-NPs we have developed a humanized mouse model of LN by engrafting peripheral blood mononuclear cells (PBMCs) from LN patients into immune-deficient NSG mice. Kidneys of these mice were highly infiltrated with human CD3+ T cells and these cells produced elevated IFNγ. Furthermore, these mice demonstrated a lower survival compared to control mice engrafted with PBMCs from healthy donors. Lastly, we tested the efficacy of the Kv1.3-NPs. We found that LN mice engrafted with PBMCs pretreated in-vitro with Kv1.3-NPs survived longer than mice engrafted with PBMCs pretreated with scramble RNA-NPs. We also found that Kv1.3-NPs were selectively delivered in-vivo into TM cells of humanized mice. Overall, these data support the potential therapeutic value of Kv1.3-NPs.

Regulation of antiviral CD8 T cell immunity by CD352/SLAMF6: from patterns to pathways

Abstract A better understanding of CD8 memory T cell (TM) formation during acute and chronic viral infections is critical for both effective immunotherapy and vaccine design. Signaling lymphocytic activation molecule family receptors (SLAMF) including CD150, CD48, CD229, CD244, CD84, CD352, and CD319 were found to be involved in T cell activation and differentiation, but their contribution to the regulation of antigen-specific T cell immunity remains incompletely understood. Here, we analyzed the dynamics of SLAMF co-expression patterns by antiviral CD8 T cells in response to acute and chronic lymphocytic choriomeningitis virus (LCMV) infection. Our results demonstrate distinct SLAMF levels not only on LCMV-specific CD8 effector (TE) and TM cell subsets, but differential kinetics during acute vs. chronic infection. Importantly, we identified CD352 as a unique marker for long-term CD8 TM potential and capacity with significant upregulation on memory-precursor (MPEC) vs. short-lived effector (SLEC) as well as on central (TCM) vs. effector memory (TEM) CD8 T cells (MPEC>>TCM>TEM≥SLEC). Moreover, in the context of chronic viral infection, we found CD352+ CD8 T cells to be partially resistant to exhaustion as demonstrated by a memory-like phenotype, preserved CD8 T cell function, and better in vivo recall capacity upon adoptive transfer into naïve hosts. Conversely, chronically-infected CD352-deficient mice showed an impaired CD8 T cell response with reduced frequencies of LCMV-specific CD8 T cells, a more exhausted phenotype, lower expression of TM markers, and reduced functional properties. Collectively, our data reveal CD352 as a potential therapeutic target to modify CD8 T cell responses during acute and chronic viral infections.

Restored and Enhanced Memory T Cell Immunity in Rheumatoid Arthritis After TNFα Blocker Treatment.

TNFα inhibitors have shaped the landscape of rheumatoid arthritis (RA) therapy with high clinical efficiency. However, their impact on T cell recall responses is not well-elucidated. We aimed to analyze the immune profiles of memory T cells in RA patients undergoing TNFα inhibitor Golimumab (GM) treatment. Frequencies of peripheral T cell subsets and cytokine expression profiles in memory T cells (TM) upon PMA/Ionomycine stimulation were determined by flow cytometry. Antigen-specific CD8 T cell immunity was analyzed through stimulating PBMCs with CMV-EBV-Flu (CEF) viral peptide pool and subsequent intracellular IFNγ staining. Both peripheral CD8 and CD4 T cells from GM treated patients had a shift pattern characterized by an enlarged effector TM and a reduced central TM cell population when compared to GM untreated group. An increase in the frequencies of TNFα+, IL-2+, and IL-17+ CD8 TM cells was observed whereas only TNFα+CD4 TM cells increased in GM treated patients. Moreover, GM treated patients contained more peripheral IFNγ-producing CD8 T cells specific to CEF viral peptides. Together, these results show a distinct T cell subset pattern and enhanced memory T cell immunity upon GM treatment, suggesting an immunoregulatory effect of TNF inhibitor Golimumab on peripheral memory T cell responses.

Establishment of human trabecular meshwork cell cultures using nontransplantable corneoscleral rims.

Human trabecular meshwork (hTM) cell isolation in academic settings utilizes the motile nature of these cells, allowing them to migrate away from the explant and proliferate on distal regions of the culture substrate. Corneoscleral rims used for transplantation are a potential source of explants for the establishment of hTM cell cultures. However, cell isolation and the initiation of primary cell cultures from ocular tissues stored in Optisol-GS medium for an extended period of time (>6 days) has proven difficult, since Optisol-GS remarkably reduces cell viability and cellularity. Therefore, explants obtained from ocular tissues stored in Optisol-GS do not often provide adequate cell yield to initiate primary cell cultures if conventional culture techniques are used. Therefore, the majority of the research on primary hTM cell isolation has been accomplished using donor tissue obtained within 72 h postmortem. The goal of this study was to develop an hTM cell isolation procedure from nontransplantable ocular materials, utilizing the anchorage dependency of TM cells. This procedure yielded functionally viable cells, eficiently dissociated from the trabecular meshwork. Isolated cells demonstrated typical hTM cell characteristics including monolayer formation, contact inhibition, phagocytosis, and responses to glucocorticoid exposure. To the best of our knowledge, this is the first time an expired explant has been utilized in the successful isolation of hTM cells. Our results clearly demonstrate the advantage of increasing the anchor points of hTM cells for enhanced cell migration out from the explants, which have limited cell proliferative capacity.

RKI-1447, a Rho kinase inhibitor, causes ocular hypotension, actin stress fiber disruption, and increased phagocytosis.

This study investigated the hypotensive effect of RKI-1447, a Rho kinase inhibitor, in a porcine ex vivo pigmentary glaucoma model. Twenty-eight porcine anterior chambers were perfused with medium supplemented with 1.67 × 107 pigment particles/ml for 48h before treatment with RKI-1447 (n = 16) or vehicle control (n = 12). Intraocular pressure (IOP) was recorded and outflow facility was calculated. Primary trabecular meshwork cells were exposed to RKI-1447 or vehicle control; effects on the cytoskeleton, motility, and phagocytosis were evaluated. Compared to baseline, the perfusion of pigment caused a significant increase in IOP in the RKI-1447 group (P = 0.003) at 48h. Subsequent treatment with RKI-1447 significantly reduced IOP from 20.14 ± 2.59 to 13.38 ± 0.91mmHg (P = 0.02). Pigment perfusion reduced the outflow facility from 0.27 ± 0.03 at baseline to 0.18 ± 0.02 at 48h (P < 0.001). This was partially reversed with RKI-1447. RKI-1447 caused no apparent histological changes in the micro- or macroscopic TM appearance. RKI-1447-treated primary TM cells showed significant disruption of the actin cytoskeleton both in the presence and absence of pigment (P < 0.001) but no effect on TM migration was observed. Pigment-treated TM cells exhibited a reduction in TM phagocytosis, which RKI-1447 reversed. RKI-1447 significantly reduces IOP by disrupting TM stress fibers and increasing TM phagocytosis. These features may make it useful for the treatment of secondary glaucomas with an increased phagocytic load.

Tat-exported peptidoglycan amidase-dependent cell division contributes to Salmonella Typhimurium fitness in the inflamed gut.

Salmonella enterica serovar Typhimurium (S. Tm) is a cause of food poisoning accompanied with gut inflammation. Although mucosal inflammation is generally thought to be protective against bacterial infection, S. Tm exploits the inflammation to compete with commensal microbiota, thereby growing up to high densities in the gut lumen and colonizing the gut continuously at high levels. However, the molecular mechanisms underlying the beneficial effect of gut inflammation on S. Tm competitive growth are poorly understood. Notably, the twin-arginine translocation (Tat) system, which enables the transport of folded proteins outside bacterial cytoplasm, is well conserved among many bacterial pathogens, with Tat substrates including virulence factors and virulence-associated proteins. Here, we show that Tat and Tat-exported peptidoglycan amidase, AmiA- and AmiC-dependent cell division contributes to S. Tm competitive fitness advantage in the inflamed gut. S. Tm tatC or amiA amiC mutants feature a gut colonization defect, wherein they display a chain form of cells. The chains are attributable to a cell division defect of these mutants and occur in inflamed but not in normal gut. We demonstrate that attenuated resistance to bile acids confers the colonization defect on the S. Tm amiA amiC mutant. In particular, S. Tm cell chains are highly sensitive to bile acids as compared to single or paired cells. Furthermore, we show that growth media containing high concentrations of NaCl and sublethal concentrations of antimicrobial peptides induce the S. Tm amiA amiC mutant chain form, suggesting that gut luminal conditions such as high osmolarity and the presence of antimicrobial peptides impose AmiA- and AmiC-dependent cell division on S. Tm. Together, our data indicate that Tat and the Tat-exported amidases, AmiA and AmiC, are required for S. Tm luminal fitness in the inflamed gut, suggesting that these proteins might comprise effective targets for novel antibacterial agents against infectious diarrhea.

Preventative tracheal administration of interleukin-27 attenuates allergic asthma by improving the lung Th1 microenvironment.

Interleukin-27 (IL-27) modulates CD4+ T-cell differentiation and function. The aim of this study is to investigate the effect and molecular mechanisms of IL-27 on the development of asthma. IL-27 was intranasally administered in an ovalbumin-induced asthma model, and lung mononuclear cells and different Th cell classes were detected by fluorescence-activated cell sorting. The effect and mechanisms of IL-27 on human bronchial epithelial (HBE) cells were investigated by measuring changes in chemotactic factors, cytokines, transcription factors, and signaling pathways. We found that intranasal administration of IL-27 could attenuate airway inflammation and hyperresponsiveness, upregulate the type 1 T helper(Th1)-T memory (Tm) cells and regulatory T (Treg) cells subgroups of lung tissue lymphocytes, and diminish the levels of type 2 T helper (Th2) cytokines. IL-27 upregulated the expression of C-C motif chemokine ligand 2 (CCL2), CCL3, and CCL4 in HBE cells and promoted the production of chemotactic factors to attract monocyte recruitment. Recruited monocytes secondarily secreted IL-27 to influence HBE cells in a positive feedback cycle. After IL-27 intervention, signal transducer and activator of transcription 1 (STAT1) phosphorylation increased, while STAT4 and STAT6 phosphorylation declined. Preventative intranasal administration of IL-27 can recruit more IL-27-secreted monocytes to the airway and change the different T-cell classes in lung. The improved Th1 environment helps to alleviate Th2-mediated allergic asthma by repairing the STAT1 pathway but not the STAT4 pathway.

Abstract 916: A screen to target EGFR (C797S) in osimertinib-resistant lung cancer identifies brigatinib-based combinations that synergistically inhibit cell growth

Abstract Up to 20% of non small-cell lung cancers are driven by activating mutations in EGFR (EGFRm). While EGFR tyrosine kinase inhibitors (TKIs) have proven highly effective in the clinic (e.g. gefitinib in first line EGFRm and osimertinib in T790M post-TKI resistance), acquired resistance mechanisms present an obstacle to curing EGFR-driven disease. To-date, approximately 20% of osimertinib-resistant cases in the 2nd line setting are EGFRm/T790M/C797S, and there is currently no approved therapy for these “triple-mutant” (TM) patients. We have obsrerved that the ALK inhibitor brigatinib shows moderate, equipotent activity against both EGFR-TM and the single activating mutant. We used this information to screen a panel of compounds to identify brigatinib-based combinations that could effectively target EGFR-TM-expressing cells. Among the most potent combination partners were selumetinib and other inhibitors of the MAP kinase pathway. These agents were able to synergistically impair growth of TM cells when combined with brigatinib in vitro, and accordingly show prolonged suppression of downstream pathway biomarkers in combination- vs. brigatinib monotherapy-treated cells. Further, we have shown effective combinations can dramatically enhance apoptotic cell death compared to brigatinib treatment alone. Critically, neither monotherapy treatment with effective combination partners, nor combinations based on osimertinib showed growth inhibition in the EGFR triple-mutant setting. Taken together, these data suggest novel therapeutic strategies for patients in this critical area of unmet need that warrant further preclinical testing. Citation Format: Matthew J. Martin, Nicolas Floc'h, Chrysiis Michaloglou, M Raymond Finlay, Richard A. Ward, Paul D. Smith, Darren A. Cross. A screen to target EGFR (C797S) in osimertinib-resistant lung cancer identifies brigatinib-based combinations that synergistically inhibit cell growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 916.

Changes In Functional Activation Of Memory T Cells Following Exercise

Memory T (TM) cells function to provide long-lasting protection against re-exposure to pathogens. The recall response of TM cells to foreign antigen is quicker and of a greater magnitude than a naïve T cell. How functional activation is altered in TM cells following a bout of exercise is not well known. PURPOSE: To quantify exercise induced changes in surface markers of early, middle, and late stage activation in memory T cells (CD4+CD45RO+CD45RA−) obtained from human subjects. METHODS: Utilizing a cross over design, untrained subjects completed a control and exercise visit. The control visit consisted of 30 min of seated rest while the exercise session entailed 3 sets x 10 reps squat at 70% 1-RM, 3x10 leg press at 70% 1-RM, and 3x10 leg extensions at 70% 1-RM with 2 min rest between sets. Venous blood samples were obtained pre and post each visit. CD4+ T cell isolation from peripheral blood was conducted through negative selection using a Human CD4+ T cell enrichment kit. CD4+ T cells were plated at 1.5 x 106 cells/ml in 200 μl of ImmunoCult T-cell expansion media directly after isolation and costimulated through CD3+CD28 or no stimulation. Cells were incubated for 1 and 3 d at 37°C in a humidified incubator with 5% CO2 and then analyzed by flow cytometry. Early (CD69), middle (CD25), and late (HLA-DR) markers of activation within the CD45RO+CD45RA− subset were quantified at days 0, 1, and 3. Data were analyzed using two-way RMANOVAs. RESULTS: There were no significant differences in any markers of activation at the pre measure (p > .05). Preliminary data suggests exercise does not alter functional activation in non-stimulated CD45RO+CD45RA− cells. There does appear to be a functional impact related to the TM cells ability to respond to stimuli post-exercise with two-fold increases observed in HLA-DR expression for cells co-stimulated through CD3+CD28. CONCLUSIONS: Exercise-induced alterations in functional activation of TM cells will need to be better quantified to determine not only the magnitude of change, but also to identify a kinetic profile of marker expression. Quantification of changes in this subset of cells will aid in our understanding how immune responses following vaccination are affected by exercise stress. Supported by an award through the Dr. George F. Haddix President’s Faculty Research Fund at Creighton University.

Infusion of donor-derived CD8+ memory T cells for relapse following allogeneic hematopoietic cell transplantation

AbstractMurine models showed that CD8+CD44hi memory T (TM) cells could eradicate malignant cells without inducing graft-versus-host disease (GVHD). We evaluated the feasibility and safety of infusing freshly isolated and purified donor-derived phenotypic CD8+ TM cells into adults with disease relapse after allogeneic hematopoietic cell transplantation (HCT). Phenotypic CD8 TM cells were isolated after unmobilized donor apheresis using a tandem immunomagnetic selection strategy of CD45RA depletion followed by CD8+ enrichment. Fifteen patients received CD8+ TM cells at escalating doses (1 × 106, 5 × 106, or 10 × 106 cells per kg). Thirteen received cytoreduction before CD8+ TM cell infusion, and 9 had active disease at the time of infusion. Mean yield and purity of the CD8+ TM infusion were 38.1% and 92.8%, respectively; >90% had CD8+ T effector memory phenotype, cytokine expression, and secretion profile. No adverse infusional events or dose-limiting toxicities occurred; GVHD developed in 1 patient (grade 2 liver). Ten patients (67%) maintained or achieved response (7 complete response, 1 partial response, 2 stable disease) for at least 3 months after infusion; 4 of the responders had active disease at the time of infusion. With a median follow-up from infusion of 328 days (range, 118-1328 days), median event-free survival and overall survival were 4.9 months (95% confidence interval [CI], 1-19.3 months) and 19.6 months (95% CI, 5.6 months to not reached), respectively. Collection and enrichment of phenotypic CD8+ TM cells is feasible, well tolerated, and associated with a low incidence of GVHD when administered as a manipulated infusion of donor lymphocytes in patients who have relapsed after HCT. This trial was registered at www.clinicaltrials.gov as #NCT01523223.

A critical epitope in CD147 facilitates memory CD4+ T-cell hyper-activation in rheumatoid arthritis

The abnormal activation of CD4+CD45RO+ memory T (Tm) cells plays an important role in the pathogenesis of rheumatoid arthritis (RA). Previous studies have shown that CD147 participates in T-cell activation. However, it remains unclear whether CD147 is involved in abnormal Tm-cell activation in RA patients. In this study, we demonstrated that CD147 was predominantly upregulated in Tm cells derived from RA patients. The anti-CD147 mAb 5A12 specifically inhibited Tm-cell activation and proliferation and further restrained osteoclastogenesis. Using a structural–functional approach, we depicted the interface between 5A12 and CD147. This allowed us to identify two critical residues, Lys63 and Asp65, as potential targets for RA treatment, as the double mutation K63A/D65A inhibited Tm-cell activation, mimicking the neutralization by 5A12. This study provides not only a theoretical basis for a “CD147-Tm/Osteoclast-RA chain” for the potential prevention and treatment of RA or other T-cell-mediated autoimmune diseases but also a new target for related drug design and development.

Extracellular vesicles have variable dose-dependent effects on cultured draining cells in the eye.

The role of extracellular vesicles (EVs) as signal mediators has been described in many biological fields. How many EVs are needed to deliver the desired physiological signal is yet unclear. Using a normal trabecular meshwork (NTM) cell culture exposed to non‐pigmented ciliary epithelium (NPCE)–derived EVs, a relevant model for studying the human ocular drainage system, we addressed the EVs dose–response effects on the Wnt signaling. The objective of the study was to investigate the dosing effects of NPCE‐derived EVs on TM Wnt signaling. EVs were isolated by PEG 8000 method from NPCE and RPE cells (used as controls) conditioned media. Concentrations were determined by Tunable Resistive Pulse Sensing method. Various exosomes concentration were incubated with TM cells, for the determination of mRNA (β‐Catenin, Axin2 and LEF1) and protein (β‐Catenin, GSK‐3β) expression using real‐time quantitative PCR and Western blot, respectively. Exposure of NTM cells for 8 hrs to low EVs concentrations was associated with a significant decreased expression of β‐Catenin, GSK‐3β, as opposed to exposure to high exosomal concentrations. Pro‐MMP9 and MMP9 activities were significantly enhanced in NTM cells treated with high EV concentrations of (X10) as compared to low EV concentrations of either NPCE‐ or RPE‐derived EVs and to untreated control. Our data support the concept that EVs biological effects are concentration‐dependent at their target site. Specifically in the present study, we described a general dose–response at the gene and MMPs activity and a different dose–response regarding key canonical Wnt proteins expression.

Adipose tissue TM cells.

Adipose tissue TM cells.

A Pck1-directed glycogen metabolic program regulates formation and maintenance of memory CD8+ T cells.

CD8+ memoryT (Tm)cells are fundamental for protective immunity against infections and cancers 1-5 . Metabolic activities are crucial in controlling memory T-cell homeostasis, but mechanisms linking metabolic signals to memory formation and survival remain elusive. Here we show that CD8+ Tm cells markedly upregulate cytosolic phosphoenolpyruvate carboxykinase (Pck1), the hub molecule regulating glycolysis, tricarboxylic acid cycle and gluconeogenesis, to increase glycogenesis via gluconeogenesis. The resultant glycogen is then channelled to glycogenolysis to generate glucose-6-phosphate and the subsequent pentose phosphate pathway (PPP) that generates abundant NADPH, ensuring high levels of reduced glutathione in Tm cells. Abrogation of Pck1-glycogen-PPP decreases GSH/GSSG ratios and increases levelsof reactive oxygen species (ROS), leading to impairment of CD8+ Tm formation and maintenance. Importantly, this metabolic regulatory mechanism could be readily translated into more efficient T-cell immunotherapy in mouse tumour models.

Clinical significance of circulating immune cells in left- and right-sided colon cancer.

BackgroundLeft-sided and right-sided colon cancers (LCCs and RCCs, respectively) differ in their epidemiology, pathogenesis, genetic and epigenetic alterations, molecular pathways and prognosis. Notably, immune response gene expression profiles have been shown to differ between patients with LCC and patients with RCC. The immune system plays an important role in tumor immunosurveillance, and there is increasing evidence that peripheral blood immune cells have a profound influence on tumor prognosis. This study aimed to determine the clinical significance of circulating immune cells with respect to colon tumor locations.MethodsDifferent types of circulating immune cells were separated and analysed based on their surface markers by flow cytometry. We compared the numbers of dendritic cells (DCs) and T cell subsets in the peripheral blood of 94 patients with RCC or LCC and analysed the proportions of these immune cells in relation to tumor stage, tumor differentiation and lymphatic metastasis.ResultsWe show that at later tumor stages, patients with LCC had higher levels of circulating myeloid DCs (P = 0.049) and plasmacytoid DCs (P = 0.018) than patients with RCC. In poorly differentiated tumors, LCC patients had significantly higher amount of plasmacytoid DCs (P = 0.036), CD4+ memory T (Tm) cells (P = 0.012), CD4+ T cells (P = 0.028), Tm cells (P = 0.014), and regulatory T cells (P = 0.001) than RCC patients. The levels of circulating CD4+ T cells, Tm cells and CD4+ Tm cells were significantly elevated at later stages in patients with LCC or RCC, while these cells decreased in poorly differentiated tumors in patients with RCC. Moreover, CD4+ Tm cell and CD4+ T cell levels are significantly associated with lymph node metastasis in patients with LCC and RCC.DiscussionCirculating immune cells were associated with tumor location, tumor stage and tumor differentiation, and can be used to predict lymphatic metastasis in patients with colon cancer. This variation in systemic immunity could contribute to the differential prognosis of patients with colon cancer.