Sublethal Irradiation Research Articles

Effects of ruxolitinib on murine regulatory T cells are immune-context dependent

Aplastic anemia is a bone marrow failure (BMF) disorder characterized by pancytopenia and hypocellular marrow from an immune-mediated etiology. Regulatory T cells (Tregs) prevent autoimmunity by suppressing autoreactive T cells. We recently demonstrated the efficacy of ruxolitinib (RUX), a JAK 1/2 inhibitor, in attenuating murine BMF. Herein, we investigated the changes of Tregs in the context of RUX treatment for murine BMF. Tregs are conventionally identified by surface expression of CD4 and CD25, in addition to intracellular transcription factor FoxP3. RUX promoted the expansion of Tregs in BMF mice defined by increased expression of FoxP3 in CD4 T cells but suppressed expression of activation marker CD25 in CD4 and CD8 T cells. In this context, CD25 is no longer a reliable surface marker for Tregs. We observed strong co-expression of FoxP3 with surface marker GITR instead of CD25 in RUX-treated BMF mice. Fluorescence-activated cell sorting (FACS)-sorted CD4+GITRhi cells showed high FoxP3 expression and intact suppressive function in vitro, suggesting GITR to be a surrogate marker for Tregs. In contrast to its expansive effect on Tregs in BMF, RUX suppressed Tregs in normal and sublethal irradiation conditions, indicating that the effects of RUX on Tregs are immune-context dependent.

Sublethal whole-body irradiation induces permanent loss and dysfunction in pathogen-specific circulating memory CD8 T cell populations

The increasing use of nuclear energy sources inevitably raises the risk of accidental or deliberate radiation exposure and associated immune dysfunction. However, the extent to which radiation exposure impacts memory CD8 T cells, potent mediators of immunity to recurring intracellular infections and malignancies, remains understudied. Using P14 CD8 T cell chimeric mice (P14 chimeras) with an lymphocytic choriomeningitis virus (LCMV) infection model, we observed that sublethal (5Gy) whole-body irradiation (WBI) induced a rapid decline in the number of naive (TN) and P14 circulating memory CD8 T cells (TCIRCM), with the former being more susceptible to radiation-induced numeric loss. While TN cell numbers rapidly recovered, as previously described, the number of P14 TCIRCM cells remained low at least 9 mo after radiation exposure. Additionally, the remaining P14 TCIRCM in irradiated hosts exhibited an inefficient transition to a central memory (CD62L+) phenotype compared to nonirradiated P14 chimeras. WBI also resulted in long-lasting T cell intrinsic deficits in memory CD8 T cells, including diminished cytokine and chemokine production along with impaired secondary expansion upon cognate Ag reencounter. Irradiated P14 chimeras displayed significantly higher bacterial burden after challenge with Listeria monocytogenes expressing the LCMV GP33-41 epitope relative to nonirradiated controls, likely due to radiation-induced numerical and functional impairments. Taken together, our findings suggest that sublethal radiation exposure caused a long-term numerical, impaired differentiation, and functional dysregulation in preexisting TCIRCM, rendering previously protected hosts susceptible to reinfection.

Spatiotemporal transcriptomic atlas reveals the dynamic characteristics and key regulators of planarian regeneration

Whole-body regeneration of planarians is a natural wonder but how it occurs remains elusive. It requires coordinated responses from each cell in the remaining tissue with spatial awareness to regenerate new cells and missing body parts. While previous studies identified new genes essential to regeneration, a more efficient screening approach that can identify regeneration-associated genes in the spatial context is needed. Here, we present a comprehensive three-dimensional spatiotemporal transcriptomic landscape of planarian regeneration. We describe a pluripotent neoblast subtype, and show that depletion of its marker gene makes planarians more susceptible to sub-lethal radiation. Furthermore, we identified spatial gene expression modules essential for tissue development. Functional analysis of hub genes in spatial modules, such as plk1, shows their important roles in regeneration. Our three-dimensional transcriptomic atlas provides a powerful tool for deciphering regeneration and identifying homeostasis-related genes, and provides a publicly available online spatiotemporal analysis resource for planarian regeneration research.

IL-6 and IL-8 secretion by human glioma cells proliferating after Gamma-knife irradiation

One of the modern methods of treating patients with primary and recurrent brain tumors is radiosurgical irradiation using Gamma Knife, which allows therapeutic doses to be delivered to tumors not exceeding 2.5 cm in diameter in 1–2 sessions. Tumor cells on the periphery of this tissue volume that receive lower radiation doses can resume proliferation and serve as a source of recurrence. The increase of radiation dose may cause necroses formation and a worsening prognosis. The properties of glioblastoma cells that survive and resume proliferation long after stereotactic irradiation are still poorly known. The aim of the work was to evaluate the expression of IL-6 and IL-8 by glioblastoma A172, R1, T2, and T98G cell lines that resumed proliferation after sublethal Gamma Knife irradiation. Cells were irradiated once at doses ranging from 6 to 16 Gy, and then cultured for 40 days. Cell number was counted weekly; lethal and sublethal irradiation doses for each glioblastoma cell line were determined. In cultures descendant from proliferation of single most resistant cells, the level of IL-6 and IL-8 secretion after 96 hours cultivation (ng/1000 cells) was determined by ELISA. The cells of all four glioblastoma lines secreted IL-6 and IL-8 into culture medium. The highest production of cytokines, never before demonstrated for glioblastomas, was discovered in R1 cells. Glioblastoma T2 also had high interleukin production levels. In contrast to these lines, glioblastoma A172 (highly sensitive to the action of cytostatic drugs and radiation) secreted IL-6 at 30 times lower level than R1 cells. Glioblastoma T98G (highly resistant to the action of cytostatic drugs and radiation) also exhibited low interleukins production level. R1, T2, and T98G glioblastoma cells that resumed proliferation after irradiation had increased secretion of IL-6 and, to a lesser extent, IL-8. The dependence of cytokine production increase on irradiation dose for these cells was not linear. In contrast, A172 cells reduced IL-6 and IL-8 secretion under irradiation. The multidirectional changes in IL-6 and IL-8 production by cells of different glioblastoma lines were long-term and persisted for more than a month. The presented results cast doubt on the possibility to use IL-6 and IL-8 production by glioblastoma cells as potential biomarkers for early diagnosis, therapy monitoring as well as prognostic markers of the disease course.

Absence of Depressive and Anxious Behavior with Genetic Dysregulation in Adult C57Bl/6J Mice after Prenatal Exposure to Ionizing Radiation.

The exposure of ionizing radiation during early gestation often leads to deleterious and even lethal effects; however, few extensive studies have been conducted on late gestational exposures. This research examined the behavior al effects of C57Bl/6J mouse offspring exposed to low dose ionizing gamma irradiation during the equivalent third trimester. Pregnant dams were randomly assigned to sham or exposed groups to either low dose or sublethal dose radiation (50, 300, or 1000 mGy) at gestational day 15. Adult offspring underwent a behavioral and genetic analysis after being raised under normal murine housing conditions. Our results indicate very little change in the behavioral tasks measuring general anxiety, social anxiety, and stress-management in animals exposed prenatally across the low dose radiation conditions. Quantitative real-time polymerase chain reactions were conducted on the cerebral cortex, hippocampus, and cerebellum of each animal; results indicate some dysregulation in markers of DNA damage, synaptic activity, reactive oxygen species (ROS) regulation, and methylation pathways in the offspring. Together, our results provide evidence in the C57Bl/6J strain, that exposure to sublethal dose radiation (<1000 mGy) during the last period of gestation leads to no observable changes in behaviour when assessed as adults, although some changes in gene expression were observed for specific brain regions. These results indicate that the level of oxidative stress occurring during late gestation for this mouse strain is not sufficient for a change in the assessed behavioral phenotype, but results in some modest dysregulation of the genetic profile of the brain.

Mitotic CDK1 and 4E-BP1 I: Loss of 4E-BP1 serine 82 phosphorylation promotes proliferative polycystic disease and lymphoma in aged or sublethally irradiated mice.

4E-BP1 is a tumor suppressor regulating cap-dependent translation that is in turn controlled by mechanistic target of rapamycin (mTOR) or cyclin-dependent kinase 1 (CDK1) phosphorylation. 4E-BP1 serine 82 (S82) is phosphorylated by CDK1, but not mTOR, and the consequences of this mitosis-specific phosphorylation are unknown. Knock-in mice were generated with a single 4E-BP1 S82 alanine (S82A) substitution leaving other phosphorylation sites intact. S82A mice were fertile and exhibited no gross developmental or behavioral abnormalities, but the homozygotes developed diffuse and severe polycystic liver and kidney disease with aging, and lymphoid malignancies after irradiation. Sublethal irradiation caused immature T-cell lymphoma only in S82A mice while S82A homozygous mice have normal T-cell hematopoiesis before irradiation. Whole genome sequencing identified PTEN mutations in S82A lymphoma and impaired PTEN expression was verified in S82A lymphomas derived cell lines. Our study suggests that the absence of 4E-BP1S82 phosphorylation, a subtle change in 4E-BP1 phosphorylation, might predispose to polycystic proliferative disease and lymphoma under certain stressful circumstances, such as aging and irradiation.

Sublethal whole-body irradiation induces permanent loss and dysfunction in pre-existing pathogen-specific memory CD8 T cell populations

Abstract The increasing use of nuclear energy sources inevitably increases the risk of accidental or deliberate radiation-exposure. However, the extent to which radiation-exposure impacts memory CD8 T cells, potent mediators of immunity to recurring intracellular infections and malignancies, remains understudied. Using P14 LCMV immune chimeric mice (P14 chimeras), we observed that sublethal (5Gy) whole body irradiation (WBI) induced a rapid decline in the number of naive (T N) and P14 circulatory memory CD8 T cells (T CIRCM) with the former being more susceptible to radiation-induced death. While T Ncell numbers rapidly recovered, the number of P14 T CIRCMcells remained low at least nine months after radiation-exposure. Additionally, the remaining P14 T CIRCMin irradiated hosts exhibited an inefficient transition to a central memory (CD62L hi) phenotype compared to non-irradiated P14 chimeras. WBI also resulted in long-lasting T-cell intrinsic deficits including diminished cytokine and chemokine production in response to antigen-stimulation along with impaired secondary expansion upon cognate Ag re-encounter. Irradiated P14 chimeras displayed significantly higher bacterial burden after challenge with L. monocytogenes expressing GP33 compared with the non-irradiated controls, likely due to radiation-induced numerical and functional impairments. Taken together, our findings suggest that sublethal radiation-exposure caused long-term numerical decline, impaired differentiation, and functional dysregulation in pre-existing T CIRCM, which rendered previously-protected hosts susceptible to re-infections. Supported by NIH Grants GM134880, AI114543, The Holden Comprehensive Cancer Center at The University of Iowa and its National Cancer Institute Award P30CA086862

Low-dose radiation followed by on-target inhibition of Galectin-3 in combination with anti-4-1BB monoclonal antibody regulates immune responses in Group 3 and Group 4 medulloblastoma mouse model (P11-13.001)

<h3>Objective:</h3> Investigation novel treatment options for Group 3 and Group 4 medulloblastoma patients <h3>Background:</h3> Group 3 and 4 medulloblastomas (MB) have a significantly worse prognosis when compared to the sonic hedgehog (SHH) or wingless (WNT) -activated subtypes and are also the most resistant to therapy. Current aggressive radiation/chemotherapy practices though improving survival, have potential long-term comorbidities with potential neurocognitive deficits impacting patients’ overall quality of life. A key factor limiting the therapeutic success of single-agent immune therapies is the poor penetration of tumor-specific immune cells into tumor microenvironments. Recent studies have demonstrated that galectin-3 (Gal-3) accelerates M2 macrophage-infiltration and restricts T cell receptor (TCR)-mediated signaling. Immunotherapy targeting CD8⁺ T cells with agonistic anti-4-1BB (CD137) monoclonal antibody (mAb) activates CD8⁺ T cells, promoting their survival and acquiring potent cytolytic properties. Building on these findings, we hypothesize that a novel treatment paradigm with immune-priming using sub-lethal low-dose radiation in combination with a Gal-3 inhibitor (anti-Gal-3 mAb) plus anti-4-1BB mAb will synergistically deplete tumor associated macrophages, restore CD8+ T cell migration, and promote autoimmune responses into tumor islets with resultant survival benefits in preclinical immunocompetent Group 3 and Group 4 MB mouse models. <h3>Design/Methods:</h3> Treatment effects were assessed using western blot, flow cytometry, H&amp;E, immunofluorescence imaging and ELISA SPOT. <h3>Results:</h3> Our data demonstrated higher levels of Gal-3 expression in Group 3 and 4 patient derived tumor tissue, as compared to non-tumor tissue. We observed that Gal-3 shifted cytokine production via switching microglia polarization to the M2 subtype. Furthermore, H&amp;E-stained tumor sections following treatment with combination low-dose radiation, Gal-3 inhibitor and anti-4-1BB mAb showed a reduction of tumor size by ~70% when compared to untreated controls. <h3>Conclusions:</h3> The results of these investigations provide critical preliminary data for further trials examining the effectiveness of immune-priming using sub-lethal low-dose radiation followed by combination treatment using a Gal-3 inhibitor plus anti-4-1BB mAb. <b>Disclosure:</b> Dr. Cachia has received personal compensation in the range of $5,000-$9,999 for serving as a Consultant for RedHill Biopharma Ltd. Dr. Cachia has received personal compensation in the range of $500-$4,999 for serving as a Consultant for GuidePoint Global LLC. Dr. Cachia has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Dedham Group. Dr. Cachia has received personal compensation in the range of $500-$4,999 for serving on a Speakers Bureau for Massachusetts Neurological Society. Dr. Eskandari has nothing to disclose. Mr. McDonald has nothing to disclose. Dr. Infinger has nothing to disclose. William A. Vandergrift has nothing to disclose. Abhay K. Varma has nothing to disclose. Sunil J. Patel has nothing to disclose. Dr. Zukas has nothing to disclose. Dr. Lindhorst has nothing to disclose. Dr. DAS has nothing to disclose.

Inflammatory signals from fatty bone marrow support DNMT3A driven clonal hematopoiesis

Both fatty bone marrow (FBM) and somatic mutations in hematopoietic stem cells (HSCs), also termed clonal hematopoiesis (CH) accumulate with human aging. However it remains unclear whether FBM can modify the evolution of CH. To address this question, we herein present the interaction between CH and FBM in two preclinical male mouse models: after sub-lethal irradiation or after castration. An adipogenesis inhibitor (PPARγ inhibitor) is used in both models as a control. A significant increase in self-renewal can be detected in both human and rodent DNMT3AMut-HSCs when exposed to FBM. DNMT3AMut-HSCs derived from older mice interacting with FBM have even higher self-renewal in comparison to DNMT3AMut-HSCs derived from younger mice. Single cell RNA-sequencing on rodent HSCs after exposing them to FBM reveal a 6-10 fold increase in DNMT3AMut-HSCs and an activated inflammatory signaling. Cytokine analysis of BM fluid and BM derived adipocytes grown in vitro demonstrates an increased IL-6 levels under FBM conditions. Anti-IL-6 neutralizing antibodies significantly reduce the selective advantage of DNMT3AMut-HSCs exposed to FBM. Overall, paracrine FBM inflammatory signals promote DNMT3A-driven clonal hematopoiesis, which can be inhibited by blocking the IL-6 pathway.

Abstract 4064: PD-1 blockade therapy enhances the antitumor effects of lymphodepletion and adoptive T cell transfer

Abstract Background: Lymphodepleting cytotoxic regimens, such as chemotherapy and radiotherapy, have shown the ability to enhance antitumor immunity. Although the mechanisms of antitumor effects augmentation in tumor-bearing hosts after lymphodepletion and T cell transfer have been intensively investigated, the influence of lymphodepletion followed by T cell transfer on immune checkpoint molecules (ICMs) expressed on tumor cells and immune cells remains to be elucidated. In the current study, we evaluated the effects of lymphodepletion and adoptive T cell transfer on ICMs in the tumor microenvironment. In addition, we investigated whether lymphodepletion and adoptive transfer of T cells augments antitumor effects of anti-PD-1 blockade therapy. Methods: B6 mice were inoculated subcutaneously with the MCA205 murine fibrosarcoma. On day 7, mice were lymphodepleted by sublethal irradiation with 500 cGy and were reconstituted intravenously with spleen cells from normal mice as a source of naïve T cells. These mice were injected intraperitoneally with anti-PD-1 mAb on days 7 and 14. On day 21, tumor tissues were harvested, and single cell suspensions were labeled with fluorophore-conjugated antibodies for FACS analyses. Results: The expressions of ICMs, including PD-L1, PD-1, TIGIT, TIM-3 and LAG-3, were upregulated on recipient CD4+ and CD8+ T cells in the tumor microenvironment. In contrast, these ICMs were significantly reduced in transferred donor T cells. Administration of anti-PD-1 antibodies after lymphodepletion and adoptive transfer of T cells significantly inhibited tumor progression. Furthermore, transfer of both donor CD4+ and CD8+ T cells was responsible for this augmentation of antitumor effects. Conclusions: Our observations suggest that a possible mechanism of the antitumor effect augmentation mediated by lymphodepletion followed by T cell transfer is the prevention of donor T cell exhaustion and dysfunction. Blockade of PD-1 significantly enhanced the antitumor effects of lymphodepletion and T cell transfer. Citation Format: Satoshi Watanabe, Miho Takahashi, Ryo Suzuki, Masashi Arita, Ko Sato, Toshiya Fujisaki, Aya Ohtsubo, Satoshi Shoji, Kunihiro Shono, Takaaki Masuda, Tomoki Sekiya, Koichiro Nozaki, Tomohiro Tanaka, Rie Kondo, Yu Saida, Satoshi Hokari, Toshiyuki Koya, Toshiaki Kikuchi. PD-1 blockade therapy enhances the antitumor effects of lymphodepletion and adoptive T cell transfer. [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 4064.

Is there Chornobyl nuclear accident signature in Scots pine radial growth and its climate sensitivity?

The extensive radioactive fallout resulting from the 1986 Chornobyl accident caused tree death near the nuclear power plant and perturbed trees communities throughout the whole Chornobyl exclusion zone. Thirty years into the post-accident period, the radiation continues to exert its fatal effects on the surviving trees. However, to what extent the continuous multi-decadal radiation exposure has affected the radial tree growth and its sensitivity to climate variation remains unascertained. In this comparative study, we measure the Scots pine radial growth and quantify its response to climate at two sites along the western track of the nuclear fallout that received significantly different doses of radiation in 1986. The common features of the two sites allow us to disentangle and intercompare the effects of sub-lethal and moderate radiation doses on the pine's growth and climatic sensitivity. We extend the response function analysis by making the first use of the Full-Duration at Half-Maximum FDHM method in dendrochronology and apply the double-moving window approach to detect the main patterns of the growth-to-climate relationships and their temporal evolution. The stand exposed to sub-lethal radiation shows a significant radial growth reduction in 1986 with a deflection period of one year. The stand exposed to moderate radiation, in contrast, demonstrates no significant decrease in growth either in 1986 or in the following years. Beyond the radiation effects, the moving response function and FDHM enabled us to detect several mutual patterns in the growth-to-climate relationships, which are seemingly unrelated to the nuclear accident. To advance our predictive understanding of the response of forest ecosystems to a massive radioactive contamination, future studies should include quantitative wood anatomy techniques.

Morphological and immunohistochemical validation personalized patient-derived xenograft model of non-small cellular lung cancer

The aim of the study was to carry out a comparative morphological and immunohistochemical analysis of peripheral non-small cell lung cancer, which served as a source of a xenograft tumor, and tissues of the third generation of a tumor that developed in animals.Material and methods. We used 19 athymic BALB/c nu/nu mice, which were intraperitoneally injected with 1×106 CD8+ after sublethal irradiation in accordance with the humanization protocol. The transplantation of a tumor obtained from a 64-year-old patient was carried out three times consecutively. Samples of the original and xenograft tumors were automatically stained with hematoxylin and eosin, rabbit anti-CK7, anti-TTF, and anti-Ki67 antibodies. The evaluation of histological samples was carried out in accordance with the WHO recommendations (2015).Results. It has been established that a third-generation tumor developing in the body of athymic humanized mice retains the morphological and immunohistochemical features of the patient's original tumor. The described approach may be used in preclinical and personalized studies in fundamental pharmacology and molecular oncology.

Non-Clinical Cell Therapy Development Using the NCG Mouse Model as a Test System.

The NCG triple immunodeficient mice on a NOD/Nju background lack functional/mature T, B, and NK cells, and have reduced macrophage and dendritic cell function. This study characterized the NCG mouse model for toxicity, engraftment and tumorigenicity assessments of cell therapies, using CD34+ hHSPC adult mobilized cells with two myeloablation regimens.Mice received sub-lethal irradiation or busulfan and were then injected intravenously with CD34+ hHSPCs (1.0 x 106 cells/mouse) or PBS (control), while positive control animals received 2 x 106 HL-60 cells/mouse. hCD34+ cell donors were treated with the mobilizing agent G-CSF prior to leukapheresis. Following injections, mouse blood samples were collected to assess engraftment rates by flow cytometry with body weights recorded periodically up to 20 weeks post-cell injection. No significant clinical signs or body weight changes were observed. At week 10 post-cell injection, the peripheral blood chimerism of hCD45+ cells was above 20%. While mCD45+ concentration was constant between week 10 and 17 in whole blood samples, hCD45+ concentration and chimerism slightly decreased at week 17. However, chimerism remained above 10%, with busulfan-treated mice presenting higher values. Chimerism was further assessed by quantifying human Alu sequences in blood and multiple organs using qPCR. Alu sequences were most abundant in the spleen and bone marrow, while lowest in the testes. In the positive control group, expected mortalities due to tumorigenesis were observed between days 27 and 40 post-cell injection. Overall, study results may be used to inform study design and potential toxicological endpoints relevant to non-clinical cell therapy development.

Accumulation of sublethal radiation damage and its effect on cell survival

Objective. Determine the extent of sublethal radiation damage (SRD) in a cell population that received a given dose of radiation and the impact of this damage on cell survival. Approach. We developed a novel formalism to account for accumulation of SRD with increasing dose. It is based on a very general formula for cell survival that correctly predicts the basic properties of cell survival curves, such as the transition from the linear-quadratic to a linear dependence at high doses. Using this formalism we analyzed extensive experimental data for photons, protons and heavy ions to evaluate model parameters, quantify the extent of SRD and its impact on cell survival. Main results. Significant accumulation of SRD begins at doses below 1 Gy. As dose increases, so does the number of damaged cells and the amount of SRD in individual cells. SRD buildup in a cell increases the likelihood of complex irrepairable damage. For this reason, during a dose fraction delivery, each dose increment makes cells more radiosensitive. This gradual radosensitization is evidenced by the increasing slope of survival curves observed experimentally. It continues until the fraction is delivered, unless radiosensitivity reaches its maximum first. The maximum radiosensitivity is achieved when SRD accumulated in most cells is the maximum damage they can repair. After this maximum is reached, the slope of a survival curve, logarithm of survival versus dose, becomes constant, dose independent. The survival curve becomes a straight line, as experimental data at high doses show. These processes are random. They cause large cell-to-cell variability in the extent of damage and radiosensitivity of individual cells. Significance. SRD is in effect a radiosensitizer and its accumulation is a significant factor affecting cell survival, especially at high doses. We developed a novel formalism to study this phenomena and reported pertinent data for several particle types.

Novel Evidence That NADPH Oxidase 2 (Nox-2) Enhances Trafficking of Hematopoietic Stem Progenitor Cells (HSPCs) By Involving ROS Mediated Activation of Nlrp3 Inflammasome

Background. NADPH oxidase 2 (Nox-2) is a superoxide-generating enzyme that forms reactive oxygen species (ROS) to regulate the redox state involved in the self-renewal of hematopoietic stem/progenitor cells (HSPCs). Moreover, ROS are released from Nox-2 expressed by cell surface and mitochondria membranes and are potent activators of intracellular pattern recognition receptor Nlrp3 inflammasome, which regulates several aspects of HSPCs biology, including their trafficking, proliferation, and metabolism. Our previous research demonstrated a novel role of Nlrp3 inflammasome in the migration, mobilization, homing, and engraftment of HSPCs (Leukemia 2022;36(1):23-32;Stem Cell Rev Rep. 2020 16(5):954-967). The role of ROS was extensively studied in the proliferation of hematopoietic cells; however, its role in the migration of HSPCs is not very well understood. Aim of the study.Based on the role of Nox-2 in activating Nlrp3 inflammasome, we hypothesized that activation of Nox-2-ROS-Nlrp3 inflammasome axis in both HSPCs and BM microenvironment orchestrates trafficking of HSPCs.Materials and Methods. First, we studied in Transwells chemotaxis of Nox-2 deficient cells to bone marrow (BM) chemoattractants, including SDF-1, S1P, and eATP. Next, to shed more light on the role of Nox2 in the trafficking of HSPCs, we performed in Nox-2-KO animals G-CSF- and AMD3100-induced pharmacological mobilization. We also studied homing and engraftment of Nox-2-KO cells in BM of wild-type (WT) animals and homing and engraftment o WT BM cells in Nox-2-KO recipients. Nox-2-KO mice were also sublethal irradiated, and we followed the recovery of peripheral blood counts in these animals. Activation of Nlrp3 inflammasome in Nox-2-KO murine Sca-1+c-kit+lineage- HSPCs was evaluated by Glow assay. To ''bypass'' Nox-2-ROS activation in control experiments, nigericin has been employed as a direct activator of Nlrp3 inflammasome. Since membrane lipid rafts (MLRs) play a crucial role optimal migration of HSPCs, we evaluated the formation of membrane lipid rafts by employing confocal analysis in Nox-2-KO cells stimulated with SDF-1. We also studied the expression of enzymes (SREBP2, HMGCs, and HMGCR) involved in providing lipid components for MLRs assemble in activated Nox-2-KO cells. Results. We noticed that Nox-2-KO animals have a profound defect in the trafficking of HSPCs as evidenced by impaired chemotaxis to BM chemoattractants, decrease in pharmacological mobilization, and defect in homing and engraftment after transplantation. Nox-2-KO mice have also defect in recovery from sublethal irradiation. At the molecular level, HSPCs from Nox-2-KO mice displayed a defect in activation of Nlrp3 inflammasome and in cholesterol and sphingolipids synthesizing enzymes, which provide lipid components required for optimal assemble of MLRs. These defects were partially ameliorated by using direct activator of Nlrp3 inflammasome that is nigericin. Conclusions. We provide for the first time evidence that Nox-2-ROS-Nlrp3 inflammasome axis operates in normal HSPCs and is crucial for regulating trafficking and post-irradiation recovery of these cells. These effects are mediated by providing components for MLRs required for optimal migration and signaling in HSPCs

Novel Evidence That Intracellular Complement Network (complosome) Is Expressed in Hematopoietic Stem Cells (HSPCs) and Regulates Their Trafficking, Metabolism, and Proliferation in an Intracrine-Dependent Manner By Engaging Pattern Recognition Receptor Nlrp3 Inflammasome

Background. Complement cascade (ComC) proteins, including C3 and C5 components, are synthesized in the liver; however, recent reports indicate their expression in lymphocytes as well (Immunity 2020;52(3):513-527). Accordingly, intracellular ComC proteins have been reported to be cleaved to active C3a and C5a mediators that interact with corresponding receptors in an intracrine-depended manner. This novel regulatory loop operating in lymphocytes has been named ''complosome''. Aim of the study.Because hematopoiesis and lymphopoiesis have a common stem cell origin and similar mechanisms are involved in regulating the biology of both lineages, we asked if complosome is also expressed and functional in normal murine hematopoietic stem progenitor cells (HSPCs). We also address a challenging question if intracellular ComC mediators could activate pattern recognition receptor Nlrp3 inflammasome that, as we reported, regulates trafficking, proliferation and metabolism of HSPCs (Leukemia 2022;36(1):23-32;Stem Cell Rev Rep. 2020 16(5):954-967). Materials and Methods. First, we phenotyped purified murine and human HSPCs for expression of major complement elements, including C3, C5, C3aR, C5aR, and cathepsin L that cleaves intracellular C3 to C3a. Next, we focused on a crucial distal executive part of ComC, protein C5, and evaluated in C5-KO and C5aR-KO mice the number of HSPCs and their clonogeneic potential. We also studied hematopoietic recovery of these animals after sublethal irradiation (650 cGy). Activation of Nlrp3 inflammasome in C5a-KO and C5aR-KO in response to hematopoietic growth factors and cytokines was evaluated by immunofluorescence Glow assay. We also studied homing and engraftment of C5-KO and C5aR-KO mice-derived HSPCs transplanted into normal WT recipients. Finally, we performed a metabolomic analysis of C5-KO, C5aR-KO, and control WT mice in steady-state conditions and after stimulation with selected hematopoietic growth factors and cytokines. Results. We show for the first time that purified murine and human HSPCs express complosome elements, including C3, C5, C3aR, C5aR, and cathepsin L. Next, C5-KO and C5aR-KO mice also have a lower number of HSPCs and clonogenic progenitors in bone marrow (BM), and display defect of hematopoietic recovery from sublethal irradiation. Furthermore, BM cells isolated from these animals had impaired homing and engraftment in normal WT recipients, supported by the observation that HSPCs from complosome deficient mice had a defect in migration to stromal-derived factor-1 (SDF-1), which correlated with decreased activation of Nlrp3 inflammasome. This defect could be alleviated by direct activation of Nlrp3 inflammasome by nigericin. Finally, our metabolic studies revealed a defect in glucose, amino acids, and lipid metabolism in C5-KO and C5aR-KO mice which correlates with complosome deficiency and Nlrp3 inflammasome activation. Conclusions. We provide for the first time evidence that complosome is expressed and functional in normal HSPCs and by engaging Nlrp3 inflammasome regulates metabolism, proliferation, and migration of HSPCs. Thus, our data sheds new intriguing light on innate immunity as a regulator of hematopoiesis that involves intracellular activation of Nlrp3 inflammasome. We currently focus on Nlrp3 inflammasome-released alarmins released from the cells in response to complosome-Nlrp3 inflammasome activation that accounts for observed phenomena. These studies are essential to develop more efficient ex vivo expansion strategies of HSPCs and to understand better intrinsic mechanisms that regulate metabolism of these cells.

Intravital Microscopy of the Native Thymus

Hematopoietic Cell Transplantation (HCT) is a common treatment for patients suffering from a variety of malignant or benign diseases, reconstituting the hematopoietic system after preconditioning treatment. In patients undergoing HCT, the capacity for the thymus to produce functional T cells is inhibited due to damage from the cytotoxic preconditioning. Endogenous thymus regeneration depends on the complex relationship between thymus stromal cells (including vascular endothelial cells (EC)) and the recruitment of de novo "seeding" thymic progenitor cells (TPCs) from the regenerated bone marrow (BM). It has been demonstrated that ECs are radio-resistant, however in the context of HCT, it is not clear which type of EC (arterial, venous, or capillary) can survive following injury. Moreover, ECs are critical cells in endogenous thymic regeneration and are involved in recruiting TPCs from the BM, but functional damage to the vascular network may alter the hemodynamics and negatively impact TPC homing and thymus regeneration. Flow cytometry, immunohistochemistry, and ex vivo imaging techniques have been applied to study the thymus in preclinical mouse models since direct visualization of the native thymus in live mice was deemed impossible. Teleost fish are an alternative model organism for direct thymus observation in vivo but redundant cytokine pathways are present which do not exist in humans and mice. Unlike fish models, in mammals T cell development critically depends on the IL-7 cytokine signaling pathway. Therefore, tools to directly visualize the mouse thymus are needed in order to study functional changes to the vascular system after cytotoxic preconditioning in an immunologically relevant preclinical animal model. We hypothesize that cytotoxic preconditioning causes functional and anatomical changes in blood vessel architecture, especially cortical vasculature, that negatively impacts TPC homing and leads to long-term changes in the thymus microenvironment. In this project, we developed intravital imaging techniques to visualize the native thymus in live mice. Using our methodology, we were able to quantify the changes to the blood vessel network after sublethal irradiation (4.5 Gy). Our results showed an increase in blood vessel diameter in mice one day after receiving irradiation. We validated this result using tissue clearing and ex vivo imaging. In addition, most cortical blood flow velocity is <500 µm/s in both control and one day after sublethal irradiation, although no significant changes were observed in blood flow velocity and shear rate between the groups at this timepoint. Taken together, our study suggests that native intravital thymus imaging is a powerful technique enabling functional and anatomical characterization of the thymus vascular network. We believe, further work will help clarify the changes to the vascular system at later timepoints and in the context of higher irradiation doses. This method enables a whole new paradigm for studying thymus biology not previously possible.

JAK 1/2 Inhibition Preserves Hematopoietic Progenitor and Stem Cells, Prevents Aplasia, Inhibits Pro-Inflammatory Cytokines, and Prolongs Survival in Murine Immune Bone Marrow Failure

Background: Immune aplastic anemia (AA) is clinically characterized by marked pancytopenia due to cytotoxic T-lymphocyte mediated hematopoietic stem and progenitor cell (HSPC) destruction. Though hematopoietic stem cell transplant and immunosuppressive therapies are effective, both are onerous to administer and a fully oral regimen is desirable. Ruxolitinib (RUX) is a Janus Kinase (JAK) 1/2 inhibitor which suppresses cytotoxic T cell activation and inhibits production of inflammatory cytokines; RUX is FDA-approved for myeloproliferative neoplasms and graft versus host disease. Methods: RUX was administered as a previously validated food additive (Rux-chow) in an immune bone marrow failure (BMF) murine model (major histocompatibility complex mismatched C57BL/6 (B6)ÞCByB6F1 lymph node (LN) cell infusion following sublethal total body irradiation (TBI)). Animals were fed Rux-chow starting at two, four, and six days after BMF induction (D+2, D+4, D+6) until day 28; a control group ate chow without RUX for the duration. Survival was assessed for 56 (D+2) or 70 (D+4, D+6) days. Blood counts, T-cells, apoptosis, and gene expression were assessed from peripheral blood, spleen or bone marrow (BM). Inflammatory cytokines implicated in AA including IFN-g, TNF-a, IL-5, IL-1a, CCL2, FasL, CCL5, and IL-10 in the plasma were measured by Luminex. Colony-forming unit (CFU) assays were performed on BM mononuclear cells mixed in semisolid methylcellulose media containing interleukin (IL)-3, IL-6, stem cell factor and erythropoietin with colonies counted at day 10; results were compared with both untreated BMF and TBI controls. Results: All RUX-treated BMF mice (days +2, +4, and +6) achieved long term survival and all untreated BMF mice died (Figure 1A). RUX rescued blood counts in BMF mice, even with delayed treatment, that were sustained with drug discontinuation. Improvement in bodyweight (BW) was seen in Rux-fed mice. Infiltration and activation of BM T-cells were suppressed with a reduction in CD4+ and CD8+ T cells, decrease in CD25+ T-cells, and an increase in splenic FOXP3+ Tregs. Gene expression in RUX treated mice showed downregulation of immune pathways including cytokine signaling in the immune system, the JAK/STAT signaling pathway, and the IFN-g pathway, indicating inhibition of T cell function. RUX therapy preserved HSPCs phenotypically and functionally: hematopoietic progenitors were significantly greater among BM cells from BMF+RUX mice, at 2 weeks and at 8 weeks following BM failure induction, compared to both untreated BMF mice and TBI controls (Figure 1B). Inflammatory cytokines IFN-g, TNF-a, IL-5, CCL-2, CCL-5, FasL, and IL-10 were suppressed on RUX at two weeks but gradually increased when drug was discontinued (except IFN-g which remained at low levels long term). Conclusion: RUX resulted in prolonged overall survival achieved by rescue of BMF as demonstrated by a sustained improvement in peripheral blood counts; this occurred even with delayed administration up to 6 days after BMF induction. RUX alleviated T-cell mediated immune destruction by suppression of T cell infiltration and activation, expansion of Tregs, as well as functional preservation of HSPCs. RUX, an orally administered medication with a favorable toxicity profile, is a potential new therapy for aplastic anemia and related syndromes. A clinical trial using RUX in patients with immune BMF is currently under development. Figures:1A: Survival with delayed RUX therapy. 1B: CFU assays in RUX treated mice, TBI controls, and BMF controls Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Ruxolitinib Stimulates Expansion of Regulatory T Cells in Murine Immune Bone Marrow Failure

Aplastic anemia (AA) is a bone marrow failure (BMF) disorder characterized by pancytopenia and hypocellular marrow. In cases that are not constitutional or due to environmental causes, an immune-mediated etiology is inferred from the efficacy of immunosuppressive therapies and extensive work implicating cytotoxic CD8+ T cells in bone marrow destruction. To study AA experimentally, BMF is induced in mice with major histocompatibility complex-mismatched C67BL/6 (B6) ⇒ CByB6F1 lymph node (LN) cell infusion following sublethal total body irradiation (TBI). Following recent FDA approval of the JAK 1/2 inhibitor ruxolitinib (RUX) for graft-versus-host disease (GVHD), and based on its suppressive effect on T cell activation and inflammatory cytokine production, we studied RUX in our murine BMF model. Our results demonstrate that RUX attenuated BMF effectively by suppressing T cell activation and proliferation. Regulatory T cells (Tregs) are a heterogeneous subpopulation of cells that prevent autoimmunity by suppressing autoreactive T cells. Murine Tregs are identified by cell surface expression of the T-cell co-receptor CD4 and the activation marker CD25 and intracellular expression of the transcription factor FoxP3. As a master regulator of Treg development, FoxP3 associates with other transcription factors to induce the Treg functional identity. When assessing Tregs in a laboratory setting, cell fixation and permeabilization are required for antibody binding to FoxP3 due to its intranuclear location, preventing isolation of viable Tregs. To isolate intact Tregs, CD4 and CD25 double positivity alone is conventional, but some prior studies evaluating JAK inhibition used only CD4+FoxP3+ and excluded CD25. Thus, there is lack of consensus in definition of these important immunoregulatory cells in different immune contexts. Our studies revealed a RUX-mediated decrease in T-cell activation, evidenced by decreased CD25 surface expression in both CD8+ [30.5±2.71 (BMF n=7) vs 9.48±1.30 (RUX, n=10), p<0.0001] and CD4+ [33.76±2.40 (BMF n=7) vs 2.29±1.08 (RUX, n=10), p<0.0001] T cells. Concurrently, FoxP3 expression was increased [7.61±2.16 (BMF n=7) vs 16.12±0.94 (RUX, n=10), p<0.01] in CD4+ T cells, indicating a higher quantity of Tregs. We therefore postulated that, in RUX treatment, CD25 expression primarily reflects activation status rather than marking induction of Tregs. We sought to identify other surrogate surface markers for FoxP3, in order to enable Treg identification and isolation to evaluate immunoregulatory function. We investigated the surface markers GITR, CD45RB, and CD127, which have been previously described in association with Tregs, to characterize Tregs in BMF control and RUX-treated BMF mice. We observed strong co-expression of GITR and FoxP3 (Figure 1A), suggesting that CD4+GITRhi expression identified the Treg phenotype. CD4+FoxP3+ cells were consistently GITRhi but had lower CD25 expression in RUX-treated BMF samples than in BMF controls, implying that changes in CD25 expression from RUX treatment may render CD25 an unreliable marker of Tregs in this context. Tregs identified by both CD4+FoxP3+ and CD4+GITRhi showed co-expression of CD45RB and CD127. The proportions of cell populations that were CD45RB and CD127 double-negative and double-positive unexpectedly differed, according to whether BMF mice were untreated or RUX-treated, suggesting possible phenotypic changes in Tregs induced by RUX. To assess the function of Tregs in the spleen of RUX-treated BMF samples, we used flow cytometry to sort Tregs based on expression of CD4+GITRhi, and we co-cultured them with activated CFSE-labeled T cells in vitro at a 1:1 ratio for 5 days. A CFSE assay demonstrated slower CFSE dilution among T cells co-cultured with Tregs compared to those without Tregs (Figure 1B). These isolated Tregs inhibited both CD4+ and CD8+ T cell proliferation, but CD8+ T cells to a greater extent, and similarly to Tregs isolated from healthy mice. Murine Tregs can be reliably identified using CD4+GITRhi in immune contexts involving altered T-cell activation. We demonstrate phenotypic changes in the proportions of Tregs expressing surface marker combinations after RUX treatment. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

IMMU-42. ADOPTIVE HEMATOPOIETIC STEM CELL AND APD1 THERAPY CONVERGE IN THE THYMUS TO PROMOTE ANTI-GLIOBLASTOMA IMMUNITY

Abstract Our lab previously published that adoptive hematopoietic stem cell (HSC) therapy administered in conjunction with aPD1 increases median survival in multiple models of CNS malignancies. Tumor-infiltrating lymphocytes (TIL) abundance is independently associated with better outcomes in many cancers. Impressively, approximately 25% of TILs are HSC-derived in early disease. Importantly, we show HSC therapy generates de novo TILs in a thymus-dependent fashion and offer evidence of altered central tolerance following aPD1 therapy, which may permit immune activation against a larger pool of tumor-associated antigens. Therapeutic HSCs were prepared from constitutive GFP-expressing (CD45.2+) and PD1KO (CD45.2+) mice and transferred into CD45.1+ hosts. Sublethal 5Gy total body irradiation was administered day 5 post-intracranial tumor implantation. The next day, 1E6 HSCs from a 1:1 – GFP:PD1KO cocktail were injected intravenously. Descriptive statistics are reported as mean ± SD. Transferred HSCs engraft, expand, differentiate, and migrate to form approximately 25% of the TIL compartment in only 15 days. We found that, 56.2% ± 20.3 of CD3+ TILs display a thymocyte-like CD4+CD8+ (DP) phenotype in early disease, and 43.8% ± 17.4 of these DP-TILs are HSC-derived. Interestingly, DP-TILs were virtually absent in advanced disease 29 days after treatment. At this stage, HSC-derived TILs most commonly fell within the single-positive (SP) TIL subsets, comprising 9.8% ± 5.6 of SP-CD8+ TILs. Notably, HSCPD1KO-derived cells were 4x more abundant amongst SP-CD8 TILs when compared to HSCGFP-derived cells (6.5% vs 1.6%, p &amp;lt; 0.05). SP-CD8 thymocytes were similarly enriched for HSCPD1KO progeny (41.1% vs 24.7%, p &amp;lt; 0.05) which may suggest PD-1 blockade directly promotes thymopoiesis. The privileged maturation of PD1-deficient HSCs is intriguingly reversed when GFP:PD1KO HSCs are co-transferred into non-tumor-bearing mice. Thus, we show the thymus is an active site where PD-1-blocking agents function, and that HSC + aPD1 therapy is uniquely positioned to leverage thymus-dependent anti-tumor immunity.