Rapid Repopulation Research Articles

Microglial repopulation restricts ocular inflammation and choroidal neovascularization in mice.

Age-related macular degeneration (AMD) is a prevalent, chronic and progressive retinal degenerative disease characterized by an inflammatory response mediated by activated microglia accumulating in the retina. In this study, we demonstrate the therapeutically effects and the underlying mechanisms of microglial repopulation in the laser-induced choroidal neovascularization (CNV) model of exudative AMD. The CSF1R inhibitor PLX3397 was used to establish a treatment paradigm for microglial repopulation in the retina. Neovascular leakage and neovascular area were examined by fundus fluorescein angiography (FFA) and immunostaining of whole-mount RPE-choroid-sclera complexes in CNV mice receiving PLX3397. Altered cellular senescence was measured by beta-galactosidase (SA-β-gal) activity and p16INK4a expression. The effect and mechanisms of repopulated microglia on leukocyte infiltration and the inflammatory response in CNV lesions were analyzed. We showed that ten days of the CSF1R inhibitor PLX3397 treatment followed by 11 days of drug withdrawal was sufficient to stimulate rapid repopulation of the retina with new microglia. Microglial repopulation attenuated pathological choroid neovascularization and dampened cellular senescence in CNV lesions. Repopulating microglia exhibited lower levels of activation markers, enhanced phagocytic function and produced fewer cytokines involved in the immune response, thereby ameliorating leukocyte infiltration and attenuating the inflammatory response in CNV lesions. The microglial repopulation described herein are therefore a promising strategy for restricting inflammation and choroidal neovascularization, which are important players in the pathophysiology of AMD.

Eradicating an invasive mammal requires local elimination and reduced reinvasion from an urban source population.

Invasive mammal eradications are increasingly attempted across large, complex landscapes. Sequentially controlled management zones can be at risk of reinvasion from adjacent uncontrolled areas, and managers must weigh the relative benefits of ensuring complete elimination from a zone or minimizing reinvasion risk. This is complicated in urban areas, where habitat heterogeneity and a lack of baseline ecological knowledge increase uncertainty. We applied a spatial agent-based model to predict the reinvasion of a well-studied species, the brushtail possum (Trichosurus vulpecula), across an urban area onto a peninsula that is the site of an elimination campaign in Aotearoa New Zealand. We represented fine-scale urban habitat heterogeneity in a land cover layer and tested management scenarios that varied four factors: the density of possums remaining following an elimination attempt, the maintenance trap density on the peninsula, and effort expended toward preventing reinvasion by means of a high-density trap buffer at the peninsula isthmus or control of the source population adjacent to the peninsula. We found that achieving complete elimination on the peninsula was crucial to avoid rapid repopulation. The urban isthmus was predicted to act as a landscape barrier and restrict immigration onto the peninsula, but reliance on this barrier alone would fail to prevent repopulation. In combination, complete elimination, buffer zone, and source population control could reduce the probability of possum repopulation to near zero. Our findings support urban landscape barriers as one tool for sequential invasive mammal elimination but reaffirm that novel methods to expose residual individuals to control will be necessary to secure elimination in management zones. Work to characterize the urban ecology of many invasive mammals is still needed.

Rodent management in Aotearoa New Zealand: approaches and challenges to landscape-scale control.

Aotearoa-New Zealand has only four rodent species, all introduced. In order of arrival, they are Pacific rat Rattus exulans, brown rat R. norvegicus, house mouse Mus musculus, and black rat R. rattus. Rodent management in New Zealand aims mainly to conserve indigenous biodiversity rather than to protect crops or manage diseases, as is usual elsewhere. We describe four major "regimes" and one major vision for rodent control in New Zealand to meet ecological restoration objectives. Current challenges for island eradications are for large islands that are remote or populated by people. Aerial 1080 is the only large-scale (tens of thousands of hectares) option for black rat control, but its application requires adjustment to counter subsequent rapid black rat repopulation. Unfenced "ecosanctuaries" (mean 720 ha) use ground-based traps and poisons to target mainly black rats and face constant reinvasion. Ecosanctuaries with mammal-resistant fences (up to 3500 ha) limit reinvasion and target more pest species and have enabled the return of previously extirpated taxa to the main islands. Predator Free 2050 aims to eradicate the rat species (but not mice) plus some other introduced mammals from New Zealand by 2050. This vision is not attainable with current tools, but research and experimental management is exploring techniques and technologies. The large scale (to 100000 ha) at which black rats are now targeted for control to extremely low abundance seems to be unique to New Zealand.

Abstract 2859: High-resolution lineage tracing for the study of cancer drug persistence at the single-cell level

Abstract Cancer drug persistence allows small fractions of otherwise drug-sensitive populations of cancer cells to survive treatment with anti-cancer drugs. Unlike drug resistance, persistence mechanisms are not driven by genetic mutations, and are thus considered reversible after a ‘drug holiday’ period. While most persistent cells remain in cell cycle arrest under drug treatment, recent work with EGFR-mutant Non-Small Cell Lung Cancer (NSCLC) cell lines has demonstrated that a small fraction of persister cells continue to undergo cell divisions under treatment with EGFR inhibitors. Thus, persister cells have a two-fold potential clinical significance: (i) they can serve as an intermediate state, allowing a non-resistant cancer cell to survive long enough and continue to divide under drug treatment to acquire a resistance mutation; and (ii) cycling persister cells might directly contribute to relapse, without a secondary resistance-driving mutation. Despite this, little is known about the underlying mechanisms by which affect the potential of a given cell to persist, or about the epigenetic traits that govern the distribution of persistence potential in a cell population. In this work, we use high-resolution lineage tracing to study lineages of drug persistent cells within a cancer cell population. Using the PC9 NSCLC cell line as a model, we demonstrate that while most lineages within the PC9 cell population can generate persisters, certain branches within the lineage tree have significantly higher persistence potentials than other branches, suggesting that inherited events determine the probability of a cell to persist under treatment. Moreover, we analyze the rate of change of the potential to persist within a single-cell-derived lineage of NSCLC cells and assess which branches within the lineage tree generate cycling persisters. Furthermore, we couple single-cell RNAseq data with lineage tracing information to identify gene expression patterns that correlate with the probability of drug-naïve cells to persist under drug treatment, allowing for insight into the cellular and molecular mechanisms underlying the continuum of the probability to persist. Finally, we demonstrate that while persistent-derived PC9 cells are indeed resensitized after a drug holiday, they give rise to a significantly higher proportion of cycling persister cells upon resumption of drug treatment, allowing for a rapid repopulation of the cell culture by cycling persisters. These results suggest that lineage tracing is a powerful tool to study drug-induced persistence and resistance and we will apply it to multiple other cell lines and tumor types. Citation Format: Benny Zhitomirsky, Jideofor Ezike, Elizabeth Hopkins, Brian P. Danysh, Laxmi Parida, Yaara Oren, Gad Getz. High-resolution lineage tracing for the study of cancer drug persistence at the single-cell level [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2859.

Putting PLX5622 into perspective: microglia in central nervous system viral infection.

Putting PLX5622 into perspective: microglia in central nervous system viral infection.

Dose-Response Analysis Describes Particularly Rapid Repopulation of Non-Small Cell Lung Cancer during Concurrent Chemoradiotherapy.

Simple SummaryDivergent results from trials of dose-escalation and acceleration suggest that optimal schedules have yet to be identified for radiotherapy of inoperable locally advanced non-small cell lung cancer. In this hypothesis-generating study radiation dose-response models were fitted to survival rates for 51 patient cohorts treated with schedules of varying total radiation dose, dose-per-fraction and duration. The best fit described repopulation running at 1.47 Gy/day for concurrent chemoradiotherapy and 0.30 Gy/day for radiotherapy alone and sequential chemoradiotherapy. The overall fitted tumour α/β ratio was 3.0 Gy. These findings imply that moderate hypofractionation of chemoradiation, within normal tissue toxicity limits, should be efficacious.(1) Purpose: We analysed overall survival (OS) rates following radiotherapy (RT) and chemo-RT of locally-advanced non-small cell lung cancer (LA-NSCLC) to investigate whether tumour repopulation varies with treatment-type, and to further characterise the low α/β ratio found in a previous study. (2) Materials and methods: Our dataset comprised 2-year OS rates for 4866 NSCLC patients (90.5% stage IIIA/B) belonging to 51 cohorts treated with definitive RT, sequential chemo-RT (sCRT) or concurrent chemo-RT (cCRT) given in doses-per-fraction ≤3 Gy over 16–60 days. Progressively more detailed dose-response models were fitted, beginning with a probit model, adding chemotherapy effects and survival-limiting toxicity, and allowing tumour repopulation and α/β to vary with treatment-type and stage. Models were fitted using the maximum-likelihood technique, then assessed via the Akaike information criterion and cross-validation. (3) Results: The most detailed model performed best, with repopulation offsetting 1.47 Gy/day (95% confidence interval, CI: 0.36, 2.57 Gy/day) for cCRT but only 0.30 Gy/day (95% CI: 0.18, 0.47 Gy/day) for RT/sCRT. The overall fitted tumour α/β ratio was 3.0 Gy (95% CI: 1.6, 5.6 Gy). (4) Conclusion: The fitted repopulation rates indicate that cCRT schedule durations should be shortened to the minimum in which prescribed doses can be tolerated. The low α/β ratio suggests hypofractionation should be efficacious.

Compensation for radiotherapy treatment interruptions due to a cyberattack: An isoeffective DVH‐based dose compensation decision tool

Unscheduled interruptions to radiotherapy treatments lead to decreased tumor control probability (TCP). Rapid cell repopulation in the tumor increases due to the absence of radiation dose, resulting in the loss of TCP. Compensation for this loss is required to prevent or reduce an extension of the patient's overall treatment time and regain the original TCP. The cyberattack on the Irish public health service in May 2021 prevented radiotherapy treatment delivery resulting in treatment interruptions of up to 12 days. Current standards for treatment gap calculations are performed using the Royal College of Radiologists (RCR) methodology, using a point‐dose for planning target volume (PTV) and the organs at risk (OAR).An in‐house tool, named EQD2VH, was created in Python to perform treatment gap calculations using the dose–volume histogram (DVH) information in DICOM data extracted from commercial treatment planning system plans. The physical dose in each dose bin was converted into equivalent dose in 2‐Gy fractions (EQD2), accounting for tumor cell repopulation. This EQD2‐based DVH provides a 2D representation of the impact of treatment gap compensation strategies on both PTV and OAR dose distributions compared to the intended prescribed treatment plan. This additional information can aid clinicians’ choice of compensation options. EQD2VH was evaluated using five high‐priority patients experiencing a treatment interruption when the cyberattack occurred. Compensation plans were created using the RCR methodology to evaluate EQD2VH as a decision‐making tool.The EQD2VH method demonstrated that the comparison of compensated treatment plans alongside the original intended treatment plans using isoeffective DVH analysis can be achieved. It enabled a visual and quantitative comparison between treatment plan options and provided an individual analysis of each structure in a patient's plan. It demonstrated potential to be a useful decision‐making tool for finding a balance between optimizing dose to PTV while protecting OARs.

Persister state-directed transitioning and vulnerability in melanoma

Melanoma is a highly plastic tumor characterized by dynamic interconversion of different cell identities depending on the biological context. Melanoma cells with high expression of the H3K4 demethylase KDM5B (JARID1B) rest in a slow-cycling, yet reversible persister state. Over time, KDM5Bhigh cells can promote rapid tumor repopulation with equilibrated KDM5B expression heterogeneity. The cellular identity of KDM5Bhigh persister cells has not been studied so far, missing an important cell state-directed treatment opportunity in melanoma. Here, we have established a doxycycline-titratable system for genetic induction of permanent intratumor expression of KDM5B and screened for chemical agents that phenocopy this effect. Transcriptional profiling and cell functional assays confirmed that the dihydropyridine 2-phenoxyethyl 4-(2-fluorophenyl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-hexa-hydro-quinoline-3-carboxylate (termed Cpd1) supports high KDM5B expression and directs melanoma cells towards differentiation along the melanocytic lineage and to cell cycle-arrest. The high KDM5B state additionally prevents cell proliferation through negative regulation of cytokinetic abscission. Moreover, treatment with Cpd1 promoted the expression of the melanocyte-specific tyrosinase gene specifically sensitizing melanoma cells for the tyrosinase-processed antifolate prodrug 3-O-(3,4,5-trimethoxybenzoyl)-(–)-epicatechin (TMECG). In summary, our study provides proof-of-concept for a dual hit strategy in melanoma, in which persister state-directed transitioning limits tumor plasticity and primes melanoma cells towards lineage-specific elimination.

Comparison of the Locoregional Outcome, Toxicities, and Compliance between Hypofractionated and Conventional Chemoradiotherapy for Head-and-Neck Cancers

Introduction: Head-and-neck cancer (HNC) treatments are elusive, and the hunt for an appropriate radiation strategy continues. Hypofractionation has the potential to provide several advantages, including a shorter overall duration that reduces rapid repopulation, dosage escalation with a higher biologically effective dose, and patient convenience. Hypofractionation is also beneficial in minimizing the danger of catching an infectious agent by reducing the number of hospital visits during the height of the COVID-19 epidemic. Materials and Methods: Between January 2020 and August 2021, 120 patients with squamous cell carcinoma of the head-and-neck subsites were randomly allocated to either the hypofractionated arm A (n = 60) or the standard fractionation arm B (n = 60) with concomitant treatment. Results: Locoregional tumor response, acute and late toxicity, and compliance were the study's endpoints. The normal tissue toxicities of each patient undergoing radiation were monitored weekly. Clinical and radiographic evaluations of locoregional control were conducted. Conclusion: Hypofractionation effectively overcomes tumor repopulation in rapidly growing tumors such as HNC, and we conclude in our study that the hypofractionated chemoradiation schedule appears to be more efficacious, with relatively superior locoregional control when compared to conventional chemoradiation with comparable normal tissue toxicities and compliance.

Rituximab induces rapid blood repopulation by CLL cells mediated through their release from immune niches and complement exhaustion

The in vivo rituximab effects in B cell malignancies are only partially understood. Here we analyzed in a large chronic lymphocytic leukemia (CLL) cohort (n = 80) the inter-patient variability in CLL cell count reduction within the first 24 h of rituximab administration in vivo, and a phenomenon of blood repopulation by malignant cells after anti-CD20 antibody therapy. Larger CLL cell elimination after rituximab infusion was associated with lower pre-therapy CLL cell counts, higher CD20 levels, and the non-exhausted capacity of complement-dependent cytotoxicity (CDC). The absolute amount of cell-surface CD20 molecules (CD20 density x CLL lymphocytosis) was a predictor for complement exhaustion during therapy. We also describe that a highly variable decrease in CLL cell counts at 5 h (88 %–2%) following rituximab infusion is accompanied in most patients by peripheral blood repopulation with CLL cells at 24 h, and in ∼20 % of patients, this resulted in CLL counts higher than before therapy. We provide evidence that CLL cells recrudescence is linked with i) CDC exhaustion, which leads to the formation of an insufficient amount of membrane attack complexes, likely resulting in temporary retention of surviving rituximab-opsonized cells by the mononuclear-phagocyte system (followed by their release back to blood), and ii) CLL cells regression from immune niches (CXCR4dimCD5bright intraclonal subpopulation). Patients with major peripheral blood CLL cell repopulation exhibited a longer time-to-progression after chemoimmunotherapy compared to patients with lower or no repopulation, suggesting chemotherapy vulnerability of CLL cells that repopulate the blood.

The role of P2Y12 in the kinetics of microglial self-renewal and maturation in the adult visual cortex in vivo

Microglia are the brain's resident immune cells with a tremendous capacity to autonomously self-renew. Because microglial self-renewal has largely been studied using static tools, its mechanisms and kinetics are not well understood. Using chronic in vivo two-photon imaging in awake mice, we confirm that cortical microglia show limited turnover and migration under basal conditions. Following depletion, however, microglial repopulation is remarkably rapid and is sustained by the dynamic division of remaining microglia, in a manner that is largely independent of signaling through the P2Y12 receptor. Mathematical modeling of microglial division demonstrates that the observed division rates can account for the rapid repopulation observed in vivo. Additionally, newly born microglia resemble mature microglia within days of repopulation, although morphological maturation is different in newly born microglia in P2Y12 knock out mice. Our work suggests that microglia rapidly locally and that newly born microglia do not recapitulate the slow maturation seen in development but instead take on mature roles in the CNS.

Home and Away: Drivers and Perceptions of Migration Among Urban Migrants and their Rural Families in the Lower Mekong River Basin of Cambodia

As the world becomes increasingly urbanized, cities across Asia continue to emerge and expand. Cambodian cities reflect this trend with rapid growth due principally to significant and continuing in-country migration. However, following the forced evacuation of cities during the Khmer Rouge regime in 1975, and subsequent rapid repopulation in the 1980’s, Cambodia’s urbanization is more recent than other Asian countries and, as a result, less investigated. This study examines rural-to-urban migration in five southern provinces around Phnom Penh—the capital and largest city in Cambodia—from the perspective of urban migrants and their rural family members. We examine how economic, environmental, and socio-cultural factors influence migrants’ current and desired movements, rural livelihoods, and the permanency of urban migration. While finding evidence to support three major theories of migration—income-differentials, environmental change, and social networks—we argue that none of these alone are sufficient to understand current migration patterns or urban migrants’ desire to return to their home village. We argue that explanations of Cambodian migration must account for the powerful, socio-cultural attraction of one’s home village and kinship, as well as the inseparability of two factors specific to provinces around Phnom Penh: the proliferation of access to microfinance and the rise of the garment industry. When these dimensions are considered, distinct patterns of migration become legible with significant implications for the viability of microfinance-backed adaptation strategies and rural livelihoods.

Targeting chronic and evolving neuroinflammation following traumatic brain injury to improve long-term outcomes: insights from microglial-depletion models.

Targeting chronic and evolving neuroinflammation following traumatic brain injury to improve long-term outcomes: insights from microglial-depletion models.

Membrane protein CAR promotes hematopoietic regeneration upon stress.

Adult hematopoietic stem cells (HSC) are quiescent most of the time, and how HSC switch from quiescence to proliferation following hematopoietic stress is unclear. Here we demonstrate that upon stress the coxsackievirus and adenovirus receptor CAR (also known as CXADR) is upregulated in HSC and critical for HSC entry into the cell cycle. Wild-type HSC were detected with more rapid repopulation ability than the CAR knockout counterparts. After fluorouracil treatment, CAR knockout HSC had lower levels of Notch1 expression and elevated protein level of Numb, a Notch antagonist. The Notch signaling inhibitor DAPT, dominant negative form of MAML (a transcriptional coactivator of Notch), or dominant negative mutant of LNX2 (an E3 ligase that acts on Numb and binds to CAR), all were capable of abrogating the function of CAR in HSC. We conclude that CAR activates Notch1 signaling by downregulating Numb protein expression to facilitate entry of quiescent HSC into the cell cycle during regeneration.

Anti-CD52 blocks EAE independent of PD-1 signals and promotes repopulation dominated by double-negative T cells and newly generated T and B cells.

Lymphocyte depletion using anti-CD52 antibody effectively reduces relapses of multiple sclerosis (MS). To begin to understand what mechanisms might control this outcome, we examined the effect of a murine-CD52-specific mAb on the depletion and repopulation of immune cells in mice with experimental autoimmune encephalomyelitis (EAE), a model of MS. We tested whether the tolerance-promoting receptor programmed cell death protein-1 (PD-1) is required for disease remission post anti-CD52, and found that PD-1-deficient mice with a more severe EAE were nevertheless effectively treated with anti-CD52. Anti-CD52 increased the proportions of newly generated T cells and double-negative (DN) T cells while reducing newly generated B cells; the latter effect being associated with a higher expression of CD52 by these cells. In the longer term, anti-CD52 caused substantial increases in the proportion of newly generated lymphocytes and DN T cells in mice with EAE. Thus, the rapid repopulation of lymphocytes from central lymphoid organs post anti-CD52 may limit further disease. Furthermore, these data identify DN T cells, a subset with immunoregulatory potential, as a significant hyperrepopulating subset following CD52-mediated depletion.

P23 Adult onset Still’s disease as a novel autoimmune complication of alemtuzumab therapy: a case report and review of alemtuzumab associated autoimmune disease

Abstract Background Alemtuzumab is a monoclonal antibody targeted to CD52, an antigen of uncertain function on T and B lymphocytes. Alemtuzumab is an established treatment for active relapsing and remitting multiple sclerosis (MS). However, autoimmune disease (AID) is being increasingly recognised as a highly prevalent adverse effect of alemtuzumab treatment. Methods We describe a case of adult onset Still’s disease (AOSD) developing in a patient six months after alemtuzumab therapy, highlighting the need for awareness of the risk of AID in those who develop symptoms after alemtuzumab treatment, some of which can be potentially life-threatening. Results Our patient, a 37 year old female, was admitted to hospital with a three week history of fevers up to 39ºC, a flitting macular rash, sore throat and polyarthritis, neutrophils 35.2x109/L (2-7.5), ferritin 8349 ug/L (normal 15-150), ALP 151 U/L (25-105). She had a history of relapsing and remitting multiple sclerosis, that went into remission after treatment with alemtuzumab six months previously. Despite 72 hours of broad-spectrum intravenous antibiotics, fever and symptoms were unchanged. Immunology and microbiology tests were negative. CT scan of neck, chest abdomen and pelvis were unremarkable. A diagnosis of AOSD was made. She received prednisolone at 40mg daily and symptoms rapidly settled. By day three of prednisolone, ferritin had fallen to 3238, neutrophil count had halved, and ALP normalised. She continues on slow weaning of steroids and remains asymptomatic. Conclusion Alemtuzumab was approved in 2013 as a treatment for active relapsing and remitting MS. Treatment consists of two courses of ‘induction’ treatment 12 months apart, aiming to deplete T and B lymphocytes, with subsequent lymphocyte repopulation. Without requirement for continued ‘maintainance’ treatment, efficacy of alemtuzumab persists for years. The most common alemtuzumab associated AID are thyroid disease, nephropathies and immune thrombocytopenia. However, other AID has been reported, including sarcoidosis. To date, no reports exist describing AOSD post alemtuzumab therapy. Although lymphocyte reconstitution post alemtuzumab is thought to account for its therapeutic effects, this same reconstitution is postulated to contribute to AID. Baker et. al. postulated that rapid re-population of B lymphocytes and slower re-population of regulatory T lymphocytes may trigger B cell mediated AID. Further, rapid repopulation of immature B cells post alemtuzumab is associated with raised serum B cell activating factor (BAFF), which remains elevated for at least a year post treatment. BAFF has been recognised in multiple animal studies to be associated with antibody mediated AID. The hypothesis that B cells play a key role in alemtuzumab related AID may be supported by the success of rituximab in treating 2 cases of alemtuzumab associated AID. The possible critical role of B cells in the pathogenesis of alemtuzumab related AID could indicate a future treatment strategy for such AID. Disclosures N. Narayan None. G. Mazibrada None. N. Amft None.

Fundamentals of Extracellular Matrix Biomaterial Assimilation: Effect of Suture Type on Attachment Strength and Cell Repopulation.

Background:The clinical results with extracellular matrix biomaterials are confounded by expectations of material response based on years of experience with permanent or degradable synthetic polymers. However, the remodeling or assimilation of extracellular matrix biomaterials is dictated by cell-mediated processes rather than fibrous encapsulation or hydrolytic degradation. Previously, we found that tissue adherence and revascularization were dictated by proximity with de-epithelialized host tissue. We now investigate the effects of polymer and fixation type on attachment strength and rate of cell repopulation in an intra-abdominal implant model.Methods:An intra-abdominal implant model in rats was used to probe assimilation properties at 4 weeks and 12 weeks with permanent and degradable suture types as well a combination of suture and biologic attachment (mesothelial abrasion). The mechanical strength of the attachment was measured by peel testing and the repopulation by automated cell counting of histologic sections.Results:The intensity of the biologic response was greater with degradable polymers than permanent polypropylene. Tissue attachment strength ranged from 2 to 15 N but changed in elasticity with time. The magnitude and distribution of cell repopulation was highly variable by suture type but ultimately did not affect the long-term strength of the soft tissue attachment.Conclusions:The tissue approximating polymer sutures were stretchy and of similar strength regardless of degradation rate or polymer type. The strongest attachment, most rapid repopulation of the deep matrix regions, and most uniform distribution of cells were found with the addition of biologic attachment.

Abstract # 2060 Microglia are extrinsically primed by the aged microenvironment

Microglia are responsible for propagating inflammatory signals from the periphery to the brain, where they drive the behavioral sickness response. With normal aging, these cells develop a pro-inflammatory, “primed” profile with increased expression of inflammatory mediators. A critical question is if this priming can be reversed. CSF1R antagonism results in elimination of microglia in the adult brain followed by rapid repopulation. Therefore, we hypothesized CSF1R antagonist-mediated microglial depletion in the aged brain would result in repopulation of new, unprimed microglia. Here we provide evidence that microglia in adult and aged mice were robustly eliminated following oral administration of CSF1R antagonist PLX5622. When antagonism was stopped, microglia repopulated the adult and aged brain at the same rate and efficiency with new cells no longer burdened with lipofuscin, the hallmark lipid debris of aging. However, RNA-Seq analysis of FACS-sorted microglia revealed these new microglia had the same primed mRNA profile. Moreover, RNA-Seq analysis of the brain microenvironment provided evidence of astrogliosis in aged mice independent of microglial depletion/repopulation. Lastly, peripheral immune challenge still caused an exaggerated microglial inflammatory response in the aged brain with prolonged behavioral deficits. These data are interpreted to indicate the microenvironment of the aged brain significantly influences the profile of repopulating/proliferating microglia. Taken together, aged microglia proliferate and repopulate the CNS, but the repopulating microglia still adopt a pro-inflammatory profile characteristic of aging.

ALLO-819, an Allogeneic Flt3 CAR T Therapy Possessing an Off-Switch for the Treatment of Acute Myeloid Leukemia

▪Patients with relapsed acute myeloid leukemia (AML) have poor prognosis and limited treatment options. Chimeric antigen receptor (CAR) T cells have demonstrated unprecedented clinical efficacy in hematological malignancies, leading to durable responses in heavily pretreated patients. Adoptive immunotherapies using T cells redirected against AML cells are being pursued as one option with potential curative intent. However, the development of autologous CAR T therapies presents a significant logistical and clinical challenge in a rapidly progressing disease setting such as AML due to the lag time of cell manufacturing. Additionally, harvesting sufficient numbers of healthy T cells from patients with AML may not always be possible. For these reasons the development of an off-the-shelf CAR T cell product may be of benefit. This work details the preclinical evaluation of ALLO-819, an allogeneic CAR T therapy targeting the receptor tyrosine kinase Flt3 (CD135), an AML target with high prevalence in all AML subtypes and limited expression outside of the hematopoietic tissue.To construct a Flt3 CAR, a panel of high affinity (KD values of 0.19 to 233 nM, determined at 37°C) fully-human antibodies was generated using phage display technology. Single-chain variable fragments (scFvs) recognizing different immunoglobulin domains of the extracellular region of Flt3 were inserted into second-generation CAR constructs and tested for their ability to redirect T cell specificity and effector function towards AML cells. A lead CAR exhibiting minimal tonic signaling and potent antitumor activity in orthotopic mouse models of AML (2.5x106 and 1x107 CAR T cells for Eol-1 and Molm-13, respectively) was selected for further engineering to incorporate a safety off-switch in cis. To accomplish this, short amino acid stretches mimicking epitopes for the FDA-approved antibody rituximab were inserted between the hinge and target-binding regions of the CAR. The CAR T cell phenotype and antitumor efficacy were not affected by the presence of the off-switch. In the presence of rituximab, Flt3 CAR T cells were efficiently lysed via complement-dependent cytotoxicity (~ 80 % CAR T cell depletion in 3 hours) in vitro and eliminated in peripheral blood and bone marrow of NSG mice (>100-fold and >300-fold, respectively).Allogeneic ALLO-819 Flt3 CAR T cells with a lower risk of TCR-mediated graft-versus-host disease and resistant to anti-CD52 antibody (alemtuzumab)-mediated lysis were generated by disruption of the T-cell receptor alpha chain (TRAC) and the CD52 loci using TALEN® gene-editing technology. Transient expression of TALEN® in Flt3 CAR T cells resulted in high-efficiency inactivation of both loci and had no impact on T cell phenotype or antitumor efficacy. ALLO-819 Flt3 CAR T cells co-cultured with primary AML blasts ex vivo displayed target-dependent activation, cytokine secretion and cytotoxic activity.Consistent with previous reports, we detected Flt3 expression on a subset of normal hematopoietic stem and progenitor cells (HSPCs) which also showed susceptibility to CAR T cell cytotoxicity. To evaluate off-tumor effects of Flt3 CAR T cells in vivo, NSG mice were administered T cells expressing a CAR with similar affinity to both mouse and human Flt3. Mouse-cross-reactive Flt3 CAR T cells exhibited off-tumor activity that was limited to a subset of bone marrow multipotent progenitors and correlated with antitumor efficacy. Administration of rituximab led to effective depletion of CAR T cells in peripheral blood that was followed by a rapid repopulation of HSPCs to levels observed in naïve mice.In summary, these results support the development of ALLO-819 Flt3 CAR T as a novel immunotherapy for the treatment of AML. DisclosuresSommer:Allogene Therapeutics: Employment, Equity Ownership, Patents & Royalties. Djuretic:Pfizer Inc.: Employment. Valton:Cellectis.Inc: Employment, Equity Ownership, Patents & Royalties. Nguyen:Allogene Therapeutics: Employment, Equity Ownership. Sutton:Allogene Therapeutics: Employment, Equity Ownership. Poulsen:Allogene Therapeutics: Employment, Equity Ownership. Smith:Cellectis. Inc: Employment, Patents & Royalties. Djuretic:Pfizer Inc.: Employment. Chaparro-Riggers:Pfizer Inc.: Employment, Patents & Royalties. Sasu:Allogene Therapeutics: Employment, Equity Ownership, Patents & Royalties.

Population recovery of a common vole population (Microtus arvalis) after population collapse.

Population collapses in small mammals occur naturally after natural disasters and during multi-annual population fluctuations as well as after man-made intervention such as rodent management action. Although there has been extensive previous work on patterns and mechanisms of population fluctuations and cyclicity, little is known about population recovery after collapse. In Europe, the common vole (Microtus arvalis) is the major pest species in agriculture, damaging crops, competing with livestock and potentially posing a health risk to people. In this study, we investigated population recovery, recovery mechanism and recovery time of common vole populations after artificially inducing a collapse through rodenticide application. The rodenticide treatment reduced abundance in spring (by about 90%) but not in summer. Demographic data (age, sex-ratio, breeding activity) suggest that it was mostly immigration and not reproduction that led to population recovery after collapse. The findings indicate that rodenticide treatment should be conducted in spring before the main reproductive season starts. The treatment effect was transient and lasted for about 3months before immigration offset the initial reduction in population abundance. This indicates that immigration patterns should be considered by managing vole populations at an appropriate spatial scale and frequency to prevent rapid repopulation. © 2018 Society of Chemical Industry.