Publications by authors named "Richard J Simpson"

320 Publications

Proteomic dissection of large extracellular vesicle surfaceome unravels interactive surface platform.

J Extracell Vesicles 2021 11;10(13):e12164

Molecular Proteomics, Baker Heart and Diabetes Institute, Melbourne, Victoria, 3004, Australia.

The extracellular vesicle (EV) surface proteome (surfaceome) acts as a fundamental signalling gateway by bridging intra- and extracellular signalling networks, dictates EVs' capacity to communicate and interact with their environment, and is a source of potential disease biomarkers and therapeutic targets. However, our understanding of surface protein composition of large EVs (L-EVs, 100-800 nm, mean 310 nm, ATP5F1A, ATP5F1B, DHX9, GOT2, HSPA5, HSPD1, MDH2, STOML2), a major EV-subtype that are distinct from small EVs (S-EVs, 30-150 nm, mean 110 nm, CD44, CD63, CD81, CD82, CD9, PDCD6IP, SDCBP, TSG101) remains limited. Using a membrane impermeant derivative of biotin to capture surface proteins coupled to mass spectrometry analysis, we show that out of 4143 proteins identified in density-gradient purified L-EVs (1.07-1.11 g/mL, from multiple cancer cell lines), 961 proteins are surface accessible. The surface molecular diversity of L-EVs include (i) bona fide plasma membrane anchored proteins (cluster of differentiation, transporters, receptors and GPI anchored proteins implicated in cell-cell and cell-ECM interactions); and (ii) membrane surface-associated proteins (that are released by divalent ion chelator EDTA) implicated in actin cytoskeleton regulation, junction organization, glycolysis and platelet activation. Ligand-receptor analysis of L-EV surfaceome (e.g., ITGAV/ITGB1) uncovered interactome spanning 172 experimentally verified cognate binding partners (e.g., ANGPTL3, PLG, and VTN) with highest tissue enrichment for liver. Assessment of biotin inaccessible L-EV proteome revealed enrichment for proteins belonging to COPI/II-coated ER/Golgi-derived vesicles and mitochondria. Additionally, despite common surface proteins identified in L-EVs and S-EVs, our data reveals surfaceome heterogeneity between the two EV-subtype. Collectively, our study provides critical insights into diverse proteins operating at the interactive platform of L-EVs and molecular leads for future studies seeking to decipher L-EV heterogeneity and function.
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http://dx.doi.org/10.1002/jev2.12164DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8612312PMC
November 2021

Acute exercise increases immune responses to SARS CoV-2 in a previously infected man.

Brain Behav Immun Health 2021 Dec 8;18:100343. Epub 2021 Sep 8.

School of Nutritional Sciences and Wellness, University of Arizona, Tucson, AZ, USA.

Evidence is emerging that exercise and physical activity provides protection against severe COVID-19 disease in patients infected with SARS-CoV-2, but it is not known how exercise affects immune responses to the virus. A healthy man completed a graded cycling ergometer test prior to and after SARS-CoV-2 infection, then again after receiving an adenovirus vector-based COVID-19 vaccine. Using whole blood SARS-CoV-2 peptide stimulation assays, IFN-γ ELISPOT assays, flow cytometry, viral-specific T-cell expansion assays and deep T-cell receptor (TCR) β sequencing, we found that exercise robustly mobilized highly functional SARS-CoV-2 specific T-cells to the blood compartment that recognized spike protein, membrane protein, nucleocapsid antigen and the B.1.1.7 α-variant, and consisted mostly of CD3+/CD8+ T-cells and double-negative (CD4-/CD8-) CD3 T-cells. The magnitude of SARS-CoV-2 T-cell mobilization with exercise was intensity dependent and robust when compared to T-cells recognizing other viruses (e.g. CMV, EBV, influenza). Vaccination enhanced the number of exercise-mobilized SARS-CoV-2 T-cells recognizing spike protein and the α-variant only. Exercise-mobilized SARS-CoV-2 specific T-cells proliferated more vigorously to peptide stimulation and maintained broad TCR-β diversity against SARS-CoV-2 antigens both before and after expansion. Neutralizing antibodies to SARS-CoV-2 were transiently elevated during exercise after both infection and vaccination. Finally, infection was associated with an increased metabolic demand to defined exercise workloads, which was restored to pre-infection levels after vaccination. This case study provides impetus for larger studies to determine if these immune responses to exercise can facilitate viral clearance, ameliorate symptoms of long COVID syndrome, and/or restore functional exercise capacity following SARS-CoV-2 infection.
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http://dx.doi.org/10.1016/j.bbih.2021.100343DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8423674PMC
December 2021

Alterations in Saliva and Plasma Cytokine Concentrations During Long-Duration Spaceflight.

Front Immunol 2021 24;12:725748. Epub 2021 Aug 24.

National Aeronautics and Space Administration (NASA) Johnson Space Center, Human Health and Performance Directorate, Houston, TX, United States.

Long-duration spaceflight is known to cause immune dysregulation in astronauts. Biomarkers of immune system function are needed to determine both the need for and effectiveness of potential immune countermeasures for astronauts. Whereas plasma cytokine concentrations are a well-established biomarker of immune status, salivary cytokine concentrations are emerging as a sensitive indicator of stress and inflammation. For this study, to aid in characterizing immune dysregulation during spaceflight, plasma and saliva cytokines were monitored in astronauts before, during and after long-duration spaceflight onboard the International Space Station. Blood was collected from 13 astronauts at 3 timepoints before, 5 timepoints during and 3 timepoints after spaceflight. Saliva was collected from 6 astronauts at 2 timepoints before spaceflight, 2 timepoints during and 3 timepoints following spaceflight. Samples were analyzed using multiplex array technology. Significant increases in the plasma concentration of IL-3, IL-15, IL-12p40, IFN-α2, and IL-7 were observed during spaceflight compared to before flight baseline. Significant decreases in saliva GM-CSF, IL-12p70, IL-10 and IL-13 were also observed during spaceflight as compared to compared to before flight baseline concentrations. Additionally, plasma TGFβ1 and TGFβ2 concentrations tended to be consistently higher during spaceflight, although these did not reach statistical significance. Overall, the findings confirm an hormonal dysregulation of immunity, appearing pro-inflammatory and Th1 in nature, persists during long-duration orbital spaceflight. These biomarkers may therefore have utility for monitoring the effectiveness of biomedical countermeasures for astronauts, with potential application in terrestrial research and medicine.
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http://dx.doi.org/10.3389/fimmu.2021.725748DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8422944PMC
August 2021

No Effect of Acute Eccentric Resistance Exercise on Immune Responses to Influenza Vaccination in Older Adults: A Randomized Control Trial.

Front Physiol 2021 12;12:713183. Epub 2021 Aug 12.

Department of Health and Human Performance, University of Houston, Houston, TX, United States.

Introduction: Older adults are at elevated risk for morbidity and mortality caused by influenza. Vaccination is the primary means of prophylaxis, but protection is often compromised in older adults. As resistance exercise mobilizes immune cells into muscle, it may enhance vaccination response.

Purpose: Compare antibody and cell mediated immune responses to influenza vaccination in older adults who performed eccentric resistance exercise immediately prior to vaccination to those who did not exercise.

Methods: Twenty nine resistance training-naive older adults (20 women, 73.9 ± 5.3 years) were randomized to 1 of 3 groups: vaccination in the same arm that exercised (Ex-S), vaccination in the opposite arm that exercised (Ex-Op), and seated rest (No-Ex). Exercise consisted of 10 sets of 5 eccentric unilateral repetitions at 80% of the pre-determined concentric one repetition maximum. Lateral raises were alternated with bicep curls. No-Ex sat quietly for 25 min. Following exercise or rest, all received the 2018 quadrivalent influenza vaccine (Seqirus Afluria) in the non-dominant deltoid. Antibody titers against each influenza vaccine strain were determined by hemagglutinin inhibition assays at baseline, 6-, and 24-weeks post-vaccination. Influenza-specific T cells were quantified after stimulation with the vaccine by intracellular cytokine staining.

Results: No significant group x time effects were found in antibody responses to any strain (interaction for A/H1N1: = 0.682; A/H3N2: = 0.644; B/Colorado/06/2017: = 0.262; B/Phuket/3073/2013: = 0.851). Groups did not differ in fold-increase of antibody titers 6- and 24-weeks post-vaccination. Influenza-specific T-cells did not differ between groups at any time (comparison at baseline: = 0.985; 6-weeks: = 0.889; 24 weeks: = 0.857). One subject (Ex-S) reported flu-like symptoms 18 weeks post-vaccination.

Conclusion: Acute arm eccentric exercise did not influence antibody titers or cell mediated immune responses to the influenza vaccine delivered post-exercise in older adults. More strenuous exercise may be required for exercise to act as an adjuvant. ClinicalTrials.gov Identifier: NCT03736759.
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http://dx.doi.org/10.3389/fphys.2021.713183DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8388854PMC
August 2021

Exercise and adrenergic regulation of immunity.

Brain Behav Immun 2021 10 21;97:303-318. Epub 2021 Jul 21.

University of Gießen, Department of Exercise Physiology and Sports Therapy, Gießen, Germany. Electronic address:

Exercise training has a profound impact on immunity, exerting a multitude of positive effects in indications such as immunosenescence, cancer, viral infections and inflammatory diseases. The immune, endocrine and central nervous systems work in a highly synergistic manner and it has become apparent that catecholamine signaling through leukocyte β-adrenergic receptors (β-ARs) is a key mechanism by which exercise mediates improvements in immune function to help mitigate numerous disease conditions. Central to this is the preferential mobilization and redistribution of effector lymphocytes with potent anti-viral and anti-tumor activity, their interaction with muscle-derived cytokines, and the effects of catecholamine signaling on mitochondrial biogenesis, immunometabolism and the resulting inflammatory response. Here, we review the impact of acute and chronic exercise on adrenergic regulation of immunity in the context of aging, cancer, viral infections and inflammatory disease. We also put forth our contention that exercise interventions designed to improve immunity, prevent disease and reduce inflammation should consider the catecholamine-AR signaling axis as a therapeutic target and ask whether or not the adrenergic signaling machinery can be 'trained' to improve immune responses to stress, disease or during the normal physiological process of aging. Finally, we discuss potential strategies to augment leukocyte catecholamine signaling to boost the effects of exercise on immunity in individuals with desensitized β-ARs or limited exercise tolerance.
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http://dx.doi.org/10.1016/j.bbi.2021.07.010DOI Listing
October 2021

Critical phosphorus requirements of Trifolium species: The importance of root morphology and root acclimation in response to phosphorus stress.

Physiol Plant 2021 Nov 30;173(3):1030-1047. Epub 2021 Jul 30.

CSIRO Agriculture and Food, Canberra, ACT, Australia.

Differences in root morphology and acclimation to low-phosphorus (P) soil were examined among eight legume species from the Trifolium Section Tricocephalum to understand how these root attributes determine P acquisition. Ornithopus sativus was included as a highly P-efficient benchmark species. Plants were grown as microswards in pots with five rates of P supplied in a topsoil layer to mimic uneven P distribution within a field soil profile. Topsoil and subsoil roots were harvested separately to enable measurement of the nutrient-foraging responses. Critical P requirement (lowest P supply for maximum yield) varied over a threefold range, reflecting differences in root morphology and acclimation of nutrient-foraging roots to P stress. Among the species, there was a 3.2-fold range in root length density, a 1.7-fold range in specific root length, and a 2.1-fold range in root hair length. O. sativus had the lowest critical P requirement, displayed a high root length density, the highest specific root length, and the longest root hairs. Acquisition of P from P-deficient soil was facilitated by development of a large root hair cylinder (i.e. a large root-soil interface). This, in turn, was determined by the intrinsic root morphology attributes of each genotype, and the plasticity of its root morphology response to internal P stress. Root acclimation in low-P soil by all species was mostly associated with preferential allocation of mass to nutrient-foraging roots. Only O. sativus and four of the Trifolium species adjusted specific root length beneficially, and only O. sativus increased its root hair length in low-P soil.
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http://dx.doi.org/10.1111/ppl.13500DOI Listing
November 2021

Regulatory Dendritic Cells Induced by Bendamustine Are Associated With Enhanced Flt3 Expression and Alloreactive T-Cell Death.

Front Immunol 2021 24;12:699128. Epub 2021 Jun 24.

Department of Pediatrics, University of Arizona, Tucson, AZ, United States.

The growth factor Flt3 ligand (Flt3L) is central to dendritic cell (DC) homeostasis and development, controlling survival and expansion by binding to Flt3 receptor tyrosine kinase on the surface of DCs. In the context of hematopoietic cell transplantation, Flt3L has been found to suppress graft-versus-host disease (GvHD), specifically host DCs. We previously reported that the pre-transplant conditioning regimen consisting of bendamustine (BEN) and total body irradiation (TBI) results in significantly reduced GvHD compared to cyclophosphamide (CY)+TBI. Pre-transplant BEN+TBI conditioning was also associated with greater Flt3 expression among host DCs and an accumulation of pre-cDC1s. Here, we demonstrate that exposure to BEN increases Flt3 expression on both murine bone marrow-derived DCs (BMDCs) and human monocyte-derived DCs (moDCs). BEN favors development of murine plasmacytoid DCs, pre-cDC1s, and cDC2s. While humans do not have an identifiable equivalent to murine pre-cDC1s, exposure to BEN resulted in decreased plasmacytoid DCs and increased cDC2s. BEN exposure and heightened Flt3 signaling are associated with a distinct regulatory phenotype, with increased PD-L1 expression and decreased ICOS-L expression. BMDCs exposed to BEN exhibit diminished pro-inflammatory cytokine response to LPS and induce robust proliferation of alloreactive T-cells. These proliferative alloreactive T-cells expressed greater levels of PD-1 and underwent increased programmed cell death as the concentration of BEN exposure increased. Alloreactive CD4 T-cell death may be attributable to pre-cDC1s and provides a potential mechanism by which BEN+TBI conditioning limits GvHD and yields T-cells tolerant to host antigen.
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http://dx.doi.org/10.3389/fimmu.2021.699128DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8264365PMC
June 2021

Exercise and the immune system: taking steps to improve responses to cancer immunotherapy.

J Immunother Cancer 2021 07;9(7)

Pediatrics, Immunobiology, and Nutritional Sciences, University of Arizona Medical Center - University Campus, Tucson, Arizona, USA.

The remarkable success of cancer immunotherapies has provided new hope to cancer patients. Unfortunately, a significant proportion of patients remain unable to respond to immunotherapy or maintain durable clinical responses. The lack of objective responses likely results from profound immune dysfunction often observed in patients with cancer. There is substantial evidence that exercise and physical activity can reduce incidence and improve outcomes in cancer patients. As the immune system is highly responsive to exercise, one potential avenue to improve immune function is through exercise and physical activity. A single event of dynamic exercise results in the substantial mobilization of leukocytes with increased functional capacities into the circulation. Chronic, or long-term, exercise leads to higher physical fitness in terms of greater cardiorespiratory function and/or muscle strength and endurance. High aerobic capacity, as measured by maximal oxygen uptake, has been associated with the reduction of dysfunctional T cells and improvements in the abundance of some T cell populations. To be sure, however, the mechanisms of exercise-mediated immune changes are both extensive and diverse. Here, we examine the evidence and theorize how acute and chronic exercise could be used to improve responses to cancer immunotherapies including immune checkpoint inhibitors, dendritic cell vaccines, natural killer cell therapies, and adoptive T cell therapies such as chimeric antigen receptor (CAR) T cells. Although the parameters of optimal exercise to yield defined outcomes remain to be determined, the available current data provide a compelling justification for additional human studies and clinical trials investigating the adjuvant use of exercise in immuno-oncology.
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http://dx.doi.org/10.1136/jitc-2020-001872DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8256759PMC
July 2021

Understanding extracellular vesicles.

Proteomics 2021 07 17;21(13-14):e2100126. Epub 2021 Jun 17.

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http://dx.doi.org/10.1002/pmic.202100126DOI Listing
July 2021

Is mechanical loading essential for exercise to preserve the aging immune system?

Immun Ageing 2021 Jun 5;18(1):26. Epub 2021 Jun 5.

Department of Immunology, University of Tübingen, Tübingen, Germany.

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http://dx.doi.org/10.1186/s12979-021-00238-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8178824PMC
June 2021

De novo transcriptome analysis for examination of the nutrition metabolic system related to the evolutionary process through which stick insects gain the ability of flight (Phasmatodea).

BMC Res Notes 2021 May 13;14(1):182. Epub 2021 May 13.

Database Center for Life Science (DBCLS), Joint Support-Center for Data Science Research, Research Organization of Information and Systems (ROIS), Mishima, Shizuoka, 411-8540, Japan.

Objective: Insects are the most evolutionarily successful groups of organisms, and this success is largely due to their flight ability. Interestingly, some stick insects have lost their flight ability despite having wings. To elucidate the shift from wingless to flying forms during insect evolution, we compared the nutritional metabolism system among flight-winged, flightless-winged, and flightless-wingless stick insect groups.

Results: Here, we report RNA sequencing of midgut transcriptome of Entoria okinawaensis, a prominent Japanese flightless-wingless stick insect, and the comparative analysis of its transcriptome in publicly available midgut transcriptomes obtained from seven stick insect species. A gene enrichment analysis for differentially expressed genes, including those obtained from winged vs wingless and flight vs flightless genes comparisons, revealed that carbohydrate metabolic process-related genes were highly expressed in the winged stick insect group. We also found that the expression of the mitochondrial enolase superfamily member 1 transcript was significantly higher in the winged stick insect group than in the wingless stick insect group. Our findings could indicate that carbohydrate metabolic processes are related to the evolutionary process through which stick insects gain the ability of flight.
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http://dx.doi.org/10.1186/s13104-021-05600-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8120901PMC
May 2021

Immunomodulatory Effects of Bendamustine in Hematopoietic Cell Transplantation.

Cancers (Basel) 2021 Apr 3;13(7). Epub 2021 Apr 3.

Department of Pediatrics, University of Arizona, Tucson, AZ 85721, USA.

Bendamustine (BEN) is a unique alkylating agent with efficacy against a broad range of hematological malignancies, although investigations have only recently started to delve into its immunomodulatory effects. These immunomodulatory properties of BEN in the context of hematopoietic cell transplantation (HCT) are reviewed here. Pre- and post-transplant use of BEN in multiple murine models have consistently resulted in reduced GvHD and enhanced GvL, with significant changes to key immunological cell populations, including T-cells, myeloid derived suppressor cells (MDSCs), and dendritic cells (DCs). Further, in vitro studies find that BEN enhances the suppressive function of MDSCs, skews DCs toward cDC1s, enhances Flt3 expression on DCs, increases B-cell production of IL-10, inhibits STAT3 activation, and suppresses proliferation of T- and B-cells. Overall, BEN has a broad range of immunomodulatory effects that, as they are further elucidated, may be exploited to improve clinical outcomes. As such, clinical trials are currently underway investigating new potential applications of BEN in the setting of allogeneic HCT.
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http://dx.doi.org/10.3390/cancers13071702DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8038415PMC
April 2021

Secreted midbody remnants are a class of extracellular vesicles molecularly distinct from exosomes and microparticles.

Commun Biol 2021 03 25;4(1):400. Epub 2021 Mar 25.

Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science (LIMS), La Trobe University, Melbourne, VIC, 3086, Australia.

During the final stages of cell division, newly-formed daughter cells remain connected by a thin intercellular bridge containing the midbody (MB), a microtubule-rich organelle responsible for cytokinetic abscission. Following cell division the MB is asymmetrically inherited by one daughter cell where it persists as a midbody remnant (MB-R). Accumulating evidence shows MB-Rs are secreted (sMB-Rs) into the extracellular medium and engulfed by neighbouring non-sister cells. While much is known about intracellular MB-Rs, sMB-Rs are poorly understood. Here, we report the large-scale purification and biochemical characterisation of sMB-Rs released from colon cancer cells, including profiling of their proteome using mass spectrometry. We show sMB-Rs are an abundant class of membrane-encapsulated extracellular vesicle (200-600 nm) enriched in core cytokinetic proteins and molecularly distinct from exosomes and microparticles. Functional dissection of sMB-Rs demonstrated that they are engulfed by, and accumulate in, quiescent fibroblasts where they promote cellular transformation and an invasive phenotype.
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http://dx.doi.org/10.1038/s42003-021-01882-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7994562PMC
March 2021

Haploidentical hematopoietic cell transplantation is even more advantageous during the COVID-19 pandemic.

Pediatr Transplant 2021 05 17;25(3):e14004. Epub 2021 Mar 17.

Department of Pediatrics, University of Arizona, Tucson, AZ, USA.

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http://dx.doi.org/10.1111/petr.14004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8250374PMC
May 2021

Physical activity: A coadjuvant treatment to COVID-19 vaccination?

Brain Behav Immun 2021 05 7;94:1-3. Epub 2021 Mar 7.

Faculty of Sports Sciences, Universidad Europea de Madrid, Madrid, Spain; Research Institute, Physical Activity and Health Research Group (PaHerg), Hospital 12 de Octubre ('imas12'), Madrid, Spain.

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http://dx.doi.org/10.1016/j.bbi.2021.03.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7937336PMC
May 2021

COVID-19-Considerations for the Female Athlete.

Front Sports Act Living 2021 16;3:606799. Epub 2021 Feb 16.

Faculty of Sport, Health and Applied Science, St Mary's University, Twickenham, United Kingdom.

The SARS CoV-2 virus (COVID-19) caused the whole sporting calendar to be paused. As we embark on the challenge of navigating through the return to play (RTP) process, there is a necessity to consider the needs of all athletes. This commentary specifically considers recommendations and requirements for the female athlete with a physiological emphasis during and following the COVID-19 pandemic, however, it will be relevant for any similar future scenarios that may present. It is important to acknowledge that there remain many unknowns surrounding COVID-19 and the female athlete both in the short- and long-term.
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http://dx.doi.org/10.3389/fspor.2021.606799DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7921484PMC
February 2021

The effects of normoxic endurance exercise on erythropoietin (EPO) production and the impact of selective β and non-selective β + β adrenergic receptor blockade.

Eur J Appl Physiol 2021 May 1;121(5):1499-1511. Epub 2021 Mar 1.

Laboratory of Integrated Physiology, Department of Health and Human Performance, University of Houston, 3875 Holman Street, Houston, TX, 77204, USA.

Purpose: Habitual endurance exercise results in increased erythropoiesis, which is primarily controlled by erythropoietin (EPO), yet studies demonstrating upregulation of EPO via a single bout of endurance exercise have been equivocal. This study compares the acute EPO response to 30 min of high versus 90 min of moderate-intensity endurance exercise and whether that response can be upregulated via selective adrenergic receptor blockade.

Methods: Using a counterbalanced, cross-over design, fifteen participants (age 28 ± 8) completed two bouts of running (30-min, high intensity vs 90-min, moderate intensity) matched for overall training stress. A separate cohort of fourteen participants (age 31 ± 6) completed three bouts of 30-min high-intensity cycling after ingesting the preferential β-adrenergic receptor (AR) antagonist bisoprolol, the non-preferential β + β antagonist nadolol or placebo. Venous blood was collected before, during, and after exercise, and serum EPO levels were determined by ELISA.

Results: No detectable EPO response was observed during or after high intensity running, however, in the moderate-intensity trial EPO was significantly elevated at both during-exercise timepoints (+ 6.8% ± 2.3% at 15 min and + 8.7% ± 2.2% at 60 min). No significant change in EPO was observed post-cycling or between the trials involving βAR blockade.

Conclusion: Neither training mode (running or cycling), nor beta-blockade significantly influenced the EPO response to 30 min of high-intensity exercise, however, 90 min of moderate-intensity running elevated EPO during exercise, returning to baseline immediately post-exercise. Identifying the optimal mode, duration and intensity required to evoke an EPO response to exercise may help tailor exercise prescriptions designed to maximize EPO response for both performance and clinical applications.
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http://dx.doi.org/10.1007/s00421-020-04558-4DOI Listing
May 2021

Transglutaminase-2, RNA-binding proteins and mitochondrial proteins selectively traffic to MDCK cell-derived microvesicles following H-Ras-induced epithelial-mesenchymal transition.

Proteomics 2021 07 19;21(13-14):e2000221. Epub 2021 Mar 19.

Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria, Australia.

Epithelial-mesenchymal transition (EMT) describes an evolutionary conserved morphogenic process defined by loss of epithelial characteristics and acquisition of mesenchymal phenotype, and altered patterns of intercellular communication, leading to functional changes in cell migration and invasion. In this regard, we have previously reported that oncogenic H-Ras induced EMT in Madin-Darby Canine Kidney (MDCK) cells (21D1 cells) trigger changes in the protein distribution pattern in cells, exosomes, and soluble protein factors (secretome) which modulate the tumor microenvironment. Here, we report that shed microvesicles (also termed microparticles/ectosomes) secreted from MDCK cells following oncogenic H-Ras-induced EMT (21D1-sMVs) are biochemically distinct from exosomes and parental MDCK-sMVs. The protein spectra of RNA-binding proteins and mitochondrial proteins in 21D1-sMVs differ profoundly compared to those of exosomes, likewise proteins associated with suppression of anoikis. We show that 21D1-sMVs promote cell migration, confer anchorage-independent growth, and induce EMT in parental MDCK cells. An unexpected and novel finding was the selective sorting of tissue transglutaminase-2 (TGM2) into 21D1-sMVs; there was no evidence of TGM2 in MDCK-sMVs. Prior treatment of 21D1-sMVs with neutralizing anti-TGM2 or anti-FN1 antibodies attenuates the invasive capability of fibroblasts. These finding suggest that microvesicle-associated TGM2 may play an important contributory role in the EMT process and warrants further investigation.
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http://dx.doi.org/10.1002/pmic.202000221DOI Listing
July 2021

Human myeloma cell- and plasma-derived extracellular vesicles contribute to functional regulation of stromal cells.

Proteomics 2021 07 5;21(13-14):e2000119. Epub 2021 Mar 5.

Myeloma Research Group, Australian Centre for Blood Diseases, Monash University/Alfred Health, Melbourne, Victoria, Australia.

Circulating small extracellular vesicles (sEV) represent promising non-invasive biomarkers that may aid in the diagnosis and risk-stratification of multiple myeloma (MM), an incurable blood cancer. Here, we comprehensively isolated and characterized sEV from human MM cell lines (HMCL) and patient-derived plasma (psEV) by specific EV-marker enrichment and morphology. Importantly, we demonstrate that HMCL-sEV are readily internalised by stromal cells to functionally modulate proliferation. psEV were isolated using various commercial approaches and pre-analytical conditions (collection tube types, storage conditions) assessed for sEV yield and marker enrichment. Functionally, MM-psEV was shown to regulate stromal cell proliferation and migration. In turn, pre-educated stromal cells favour HMCL adhesion. psEV isolated from patients with both pre-malignant plasma cell disorders (monoclonal gammopathy of undetermined significance [MGUS]; smouldering MM [SMM]) and MM have a similar ability to promote cell migration and adhesion, suggesting a role for both malignant and pre-malignant sEV in disease progression. Proteomic profiling of MM-psEV (305 proteins) revealed enrichment of oncogenic factors implicated in cell migration and adhesion, in comparison to non-disease psEV. This study describes a protocol to generate morphologically-intact and biologically functional sEV capable of mediating the regulation of stromal cells, and a model for the characterization of tumour-stromal cross-talk by sEV in MM.
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http://dx.doi.org/10.1002/pmic.202000119DOI Listing
July 2021

The impact of high-intensity interval exercise training on NK-cell function and circulating myokines for breast cancer prevention among women at high risk for breast cancer.

Breast Cancer Res Treat 2021 Jun 8;187(2):407-416. Epub 2021 Feb 8.

Department of Pediatrics, The University of Arizona, Tucson, AZ, USA.

Purpose: Preclinical evidence suggests that natural killer cell (NK-cell) function and myokines facilitate the protective effects of exercise for breast cancer prevention. Since higher-intensity exercise acutely promotes greater mobilization and larger changes in NK-cell cytotoxicity than lower-intensity, high-intensity interval training (HIIT) might offer increased immune protection compared to moderate-intensity continuous-training (MICT). This study compared a 12-week HIIT program to a 12-week MICT program and usual care on changes in resting NK-cell function and circulating myokines among women at high risk for breast cancer.

Methods: Thirty-three women were randomized to HIIT, MICT, or usual care, for a supervised exercise intervention. Blood was collected at baseline and end-of-study. The cytotoxic activity of CD3-/CD56+ NK-cells against the K562 target cell line in vitro was determined by flow cytometry. Circulating myokines (IL-15, IL-6, irisin, OSM, osteonectin, IL-7) were assessed with luminex multiplex assays and ELISA. One-way ANOVA and paired sample t-tests assessed between- and within-group differences, respectively. Pearson correlation coefficients determined relationships between baseline fitness and change variables.

Results: Significant differences were not observed between groups for change in NK-cell function or circulating myokines (p > 0.05). Significant correlations were only observed for baseline peak aerobic capacity (ml/kg/min) and change in NK-cell-specific lysis (r = - 0.43, p = 0.02) and hemacytotoxicity for the total sample (r = - 0.46, p = 0.01).

Conclusion: Our findings suggest that exercise intensity may not significantly impact change in resting NK-cell function and circulating myokines among women at high risk for breast cancer. Structured exercise training may have a larger impact on NK-cell function in those with lower levels of cardiorespiratory fitness.

Clinical Trial Registration: NCT02923401; Registered on October 4, 2016.
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http://dx.doi.org/10.1007/s10549-021-06111-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8189992PMC
June 2021

A Protocol for Isolation, Purification, Characterization, and Functional Dissection of Exosomes.

Methods Mol Biol 2021 ;2261:105-149

Baker Heart and Diabetes Institute, Melbourne, VIC, Australia.

Extracellular vesicles (EVs) are membrane-enclosed vesicles released by cells. They carry proteins, nucleic acids, and metabolites which can be transferred to a recipient cell, locally or at a distance, to elicit a functional response. Since their discovery over 30 years ago, the functional repertoire of EVs in both physiological (e.g., organ morphogenesis, embryo implantation) and pathological (e.g., cancer, neurodegeneration) conditions has cemented their crucial role in intercellular communication. Moreover, because the cargo encapsulated within circulating EVs remains protected from degradation, their diagnostic as well as therapeutic (such as drug delivery tool) applications have garnered vested interest. Global efforts have been made to purify EV subtypes from biological fluids and in vitro cell culture media using a variety of strategies and techniques, with a major focus on EVs of endocytic origin called exosomes (30-150 nm in size). Given that the secretome comprises of soluble secreted proteins, protein aggregates, RNA granules, and EV subtypes (such as exosomes, shed microvesicles, apoptotic bodies), it is imperative to purify exosomes to homogeneity if we are to perform biochemical and biophysical characterization and, importantly, functional dissection. Besides understanding the composition of EV subtypes, defining molecular bias of how they reprogram target cells also remains of paramount importance in this area of active research. Here, we outline a systematic "how to" protocol (along with useful insights/tips) to obtain highly purified exosomes and perform their biophysical and biochemical characterization. This protocol employs a mass spectrometry-based proteomics approach to characterize the protein composition of exosomes. We also provide insights on different isolation strategies and their usefulness in various downstream applications. We outline protocols for lipophilic labeling of exosomes to study uptake by a recipient cell, investigating cellular reprogramming using proteomics and studying functional response to exosomes in the Transwell-Matrigel™ Invasion assay.
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http://dx.doi.org/10.1007/978-1-0716-1186-9_9DOI Listing
March 2021

Analysis of Annotated and Unannotated Long Noncoding RNAs from Exosome Subtypes Using Next-Generation RNA Sequencing.

Methods Mol Biol 2021 ;2254:195-218

Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science (LIMS), La Trobe University, Melbourne, VIC, Australia.

Long noncoding RNAs (lncRNAs) contain >200 nucleotides and act as regulatory molecules in transcription and translation processes in both normal and pathological conditions. LncRNAs have been reported to localize in nuclei, cytoplasm, and, more recently, extracellular vesicles such as exosomes. Exosomal lncRNAs have gained much attention as exosomes secreted from one cell type can transfer their cargo (e.g., protein, RNA species, and lipids) to recipient cells and mediate phenotypic changes in the recipient cell. In recent years, many exosomal lncRNAs have been discovered and annotated and are attracting much attention as potential markers for disease diagnosis and prognosis. It is expected that many exosomal lncRNAs are yet to be identified. However, characterization of unannotated exosomal RNAs with non-protein-coding sequences from massive RNA sequencing data is technically challenging. Here, we describe a method for the discovery of annotated and unannotated exosomal lncRNA. This method includes a large-scale isolation and purification strategy for exosome subtypes, using the human colorectal cancer cell line (LIM1863) as a model. The method inputs RNA sequencing clean reads and performs transcript assembly to identify annotated and unannotated exosomal lncRNAs. Cutoffs (length, number of exon, classification code, and human protein-coding probability) are used to identify potentially novel exosomal lncRNAs. Raw read count calculation and differential expression analysis are also introduced for downstream analysis and candidate selection. Exosomal lncRNA candidates are validated using RT-qPCR. This method provides a template for exosomal lncRNA discovery and analysis from next-generation RNA sequencing.
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http://dx.doi.org/10.1007/978-1-0716-1158-6_12DOI Listing
March 2021

Construction of TUATinsecta database that integrated plant and insect database for screening phytophagous insect metabolic products with medicinal potential.

Sci Rep 2020 10 15;10(1):17509. Epub 2020 Oct 15.

Department of Science of Biological Production, Graduate School of Agriculture, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu-shi, Tokyo, 183-8509, Japan.

Phytophagous insect larvae feed on plants containing secondary metabolic products with biological activity against other predatory organisms. Phytophagous insects can use their specialised metabolic systems to covert these secondary metabolic products into compounds with therapeutic properties useful to mankind. Some Asians drink tea decoctions made from phytophagous insect frass which is believed to be effective against inflammatory diseases. However, insects that can convert plant-derived secondary metabolic products into useful human therapeutic agents remain poorly studied. Here, we constructed the TUATinsecta database by integrating publicly plant/insect datasets for the purpose of selecting insect species. Using TUAT-insecta we selected the Asian swallowtail butterfly, Papilio xuthus larvae fed on several species of Rutaceous plants and examined whether the plant-derived secondary metabolites, especially those present in frass, were chemically altered or not. We extracted metabolic products from frass using three organic solvents with different polarities, and evaluated solvent fractions for their cytotoxic effects against several human cell lines. We found that chloroform frass extracts from P. xuthus larvae fed on Poncirus trifoliata leaves contained significant cytotoxic activity. Our findings demonstrate that screening of insect species using the 'TUATinsecta' database provides an important pipeline for discovering novel therapeutic agents that might be useful for mankind.
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http://dx.doi.org/10.1038/s41598-020-74590-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7566601PMC
October 2020

Load Magnitude and Locomotion Pattern Alter Locomotor System Function in Healthy Young Adult Women.

Front Bioeng Biotechnol 2020 16;8:582219. Epub 2020 Sep 16.

Neuromuscular Research Laboratory and Warrior Human Performance Research Center, Department of Sports Medicine and Nutrition, University of Pittsburgh, Pittsburgh, PA, United States.

Introduction: During cyclical steady state ambulation, such as walking, variability in stride intervals can indicate the state of the system. In order to define locomotor system function, observed variability in motor patterns, stride regulation and gait complexity must be assessed in the presence of a perturbation. Common perturbations, especially for military populations, are load carriage and an imposed locomotion pattern known as forced marching (FM). We examined the interactive effects of load magnitude and locomotion pattern on motor variability, stride regulation and gait complexity during bipedal ambulation in recruit-aged females.

Methods: Eleven healthy physically active females (18-30 years) completed 1-min trials of running and FM at three load conditions: no additional weight/bodyweight (BW), an additional 25% of BW (BW + 25%), and an additional 45% of BW (BW + 45%). A goal equivalent manifold (GEM) approach was used to assess motor variability yielding relative variability (RV; ratio of "good" to "bad" variability) and detrended fluctuation analysis (DFA) to determine gait complexity on stride length (SL) and stride time (ST) parameters. DFA was also used on GEM outcomes to calculate stride regulation.

Results: There was a main effect of load ( = 0.01) on RV; as load increased, RV decreased. There was a main effect of locomotion ( = 0.01), with FM exhibiting greater RV than running. Strides were regulated more tightly and corrected quicker at BW + 45% compared ( < 0.05) to BW. Stride regulation was greater for FM compared to running. There was a main effect of load for gait complexity ( = 0.002); as load increased gait complexity decreased, likewise FM had less ( = 0.02) gait complexity than running.

Discussion: This study is the first to employ a GEM approach and a complexity analysis to gait tasks under load carriage. Reduction in "good" variability as load increases potentially exposes anatomical structures to repetitive site-specific loading. Furthermore, load carriage magnitudes of BW + 45% potentially destabilize the system making individuals less adaptable to additional perturbations. This is further evidenced by the decrease in gait complexity, which all participants demonstrated values similarly observed in neurologically impaired populations during the BW + 45% load condition.
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http://dx.doi.org/10.3389/fbioe.2020.582219DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7525027PMC
September 2020

Intrinsic root morphology determines the phosphorus acquisition efficiency of five annual pasture legumes irrespective of mycorrhizal colonisation.

Funct Plant Biol 2021 01;48(2):156-170

University of New England, School of Environmental and Rural Science, Armidale, NSW 2351, Australia.

Mycorrhizal fungi are ubiquitous in agroecosystems and form symbiotic associations that contribute to the phosphorus (P) acquisition of many plants. The impact of mycorrhizas is most pronounced in P-deficient soil and commonly involves modifications to the root morphology of colonised plants. However, the consequences of mycorrhizal colonisation on root acclimation responses to P stress are not well described. Five annual pasture legumes, with differing root morphologies, were grown to determine the effect of mycorrhizal colonisation on shoot yield, root morphology and P uptake. Micro-swards of each legume were established in pots filled with a topsoil layer that had been amended with five rates of P fertiliser. The topsoil overlaid a low-P subsoil that mimicked the stratification of P that occurs under pasture. Mycorrhizal colonisation improved P acquisition and shoot yield in the low-P soil treatments, but did not reduce the critical external P requirement of the legumes for near-maximum yield. The yield responses of the mycorrhizal plants were associated with reduced dry matter allocation to topsoil roots, which meant that the P acquisition benefit associated with mycorrhizal colonisation was not additive in the P-deficient soil. The contribution of the mycorrhizal association to P acquisition was consistent among the legumes when they were compared at an equivalent level of plant P stress, and was most pronounced below a P stress index of ~0.5. The intrinsic root morphology of the legumes determined their differences in P-acquisition efficiency irrespective of mycorrhizal colonisation.
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http://dx.doi.org/10.1071/FP20007DOI Listing
January 2021

The effects of β and β adrenergic receptor blockade on the exercise-induced mobilization and ex vivo expansion of virus-specific T cells: implications for cellular therapy and the anti-viral immune effects of exercise.

Cell Stress Chaperones 2020 11 10;25(6):993-1012. Epub 2020 Aug 10.

Laboratory of Integrated Physiology, Department of Health and Human Performance, University of Houston, Houston, TX, USA.

The adoptive transfer of donor-derived virus-specific T cells (VSTs) is an effective treatment for infections following allogeneic hematopoietic cell transplantation. Acute exercise mobilizes effector lymphocytes and VSTs to the circulation and augments the ex vivo manufacture of VSTs. This study determined if β adrenergic receptor (AR) signaling precipitated the VST response to acute exercise. Healthy participants (n = 12) completed 30 min of steady-state cycling exercise after ingesting a placebo, a β AR antagonist (nadolol) or a β AR antagonist (bisoprolol). Circulating VSTs to cytomegalovirus (CMV), Epstein-Barr virus (EBV), and adenovirus (AdV) antigens were enumerated before and after exercise, and peripheral blood mononuclear cells were cultured with viral peptides for 8 days to expand multi-VSTs. Compared with placebo, nadolol blunted the exercise-induced mobilization of CMV-VSTs (Δ VSTs/100,000 CD3 T cells = 93 ± 104 vs. 22 ± 91 for placebo and nadolol, respectively; p = 0.036), while bisoprolol did not, despite both drugs evoking similar reductions in exercising heart rate and blood pressure. Circulating AdV and EBV VSTs (VSTs/mL blood) only increased after exercise with placebo. Although not significant, nadolol partially mitigated exercise-induced increases in multi-VST expansion, particularly in participants that demonstrated an exercise-induced increase in VST expansion. We conclude that exercise-induced enhancements in VST mobilization and expansion are at least partially β AR mediated, thus highlighting a role for the β AR in targeted therapy for the augmentation of VST immune cell therapeutics in the allogeneic adoptive transfer setting. Moreover, long-term regular exercise may provide additional viral protection in the host through frequent β AR-dependent mobilization and redistribution of VSTs cumulated with each bout of exercise.
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http://dx.doi.org/10.1007/s12192-020-01136-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7591642PMC
November 2020

Bendamustine Conditioning Skews Murine Host DCs Toward Pre-cDC1s and Reduces GvHD Independently of Batf3.

Front Immunol 2020 16;11:1410. Epub 2020 Jul 16.

Department of Immunobiology, University of Arizona, Tucson, AZ, United States.

Graft-versus-host disease (GvHD) remains the second leading cause of death in allogeneic hematopoietic stem cell transplantation recipients, highlighting the need for improved preventative strategies. Our laboratory has previously demonstrated in an experimental bone marrow transplantation (BMT) model that bendamustine combined with total body irradiation (BEN+TBI) is a safer alternative to cyclophosphamide with TBI (CY+TBI). The biological mechanisms of action of BEN have not been fully elucidated and likely involve multiple cell populations. Host dendritic cells (DCs) can prime naïve donor T-cells immediately following transplantation, making host DCs critical for the initiation phase of GvHD. We hypothesized that BEN+TBI conditioning favorably alters host DC composition to reduce GvHD. We demonstrate that host DCs treated with BEN+TBI induce less allogeneic T-cell proliferation than those conditioned with CY+TBI. We further show that BEN+TBI conditioning results in greater total numbers of all host DC subsets but with a more favorable composition compared to CY+TBI with significantly larger proportions of type 1 conventional DCs (cDC1), a highly regulatory DC subset capable of suppressing GvHD. Our studies using recipient Batf3 KO mice indicate that CD8α+ cDC1s are largely dispensable for the reduced GvHD following BEN+TBI conditioning. We found a higher frequency of host pre-cDC1s with BEN+TBI conditioning in both wild-type (WT) and Batf3 KO mice, which was inversely associated with GvHD. Additionally, we observed that BEN treatment results in greater expression of Flt3 receptor (CD135) on host DCs compared to CY, potentially contributing to the skewing of host DCs toward cDC1s. Further, BEN+TBI conditioning results in host cDCs with greater expression of PIR-B, an inhibitory receptor capable of preventing lethal GvHD. We conclude that BEN+TBI is a safer alternative to CY+TBI, resulting in a greater frequency of host pre-cDC1s and limiting GvHD.
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http://dx.doi.org/10.3389/fimmu.2020.01410DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7378358PMC
April 2021

Exosomes Derived from the Human Primary Colorectal Cancer Cell Line SW480 Orchestrate Fibroblast-Led Cancer Invasion.

Proteomics 2020 07;20(14):e2000016

Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria, 3086, Australia.

In localized tumors, basement membrane (BM) prevents invasive outgrowth of tumor cells into surrounding tissues. When carcinomas become invasive, cancer cells either degrade BM or reprogram stromal fibroblasts to breach BM barrier and lead invasion of cancer cells into surrounding tissues in a process called fibroblast-led invasion. However, tumor-derived factors orchestrating fibroblast-led invasion remain poorly understood. Here it is shown that although early-stage primary colorectal adenocarcinoma (SW480) cells are themselves unable to invade Matrigel matrix, they secrete exosomes that reprogram normal fibroblasts to acquire de novo capacity to invade matrix and lead invasion of SW480 cells. Strikingly, cancer cells follow leading fibroblasts as collective epithelial-clusters, thereby circumventing need for epithelial to mesenchymal transition, a key event associated with invasion. Moreover, acquisition of pro-invasive phenotype by fibroblasts treated with SW480-derived exosomes relied on exosome-mediated MAPK pathway activation. Mass spectrometry-based protein profiling reveals that cancer exosomes upregulate fibroblasts proteins implicated in focal adhesion (ITGA2/A6/AV, ITGB1/B4/B5, EGFR, CRK), regulators of actin cytoskeleton (RAC1, ARF1, ARPC3, CYFIP1, NCKAP1, ICAM1, ERM complex), and signalling pathways (MAPK, Rap1, RAC1, Ras) important in pro-invasive remodeling of extracellular matrix. Blocking tumor exosome-mediated signaling to fibroblasts therefore represents an attractive therapeutic strategy in restraining tumors by perturbing stroma-driven invasive outgrowth.
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http://dx.doi.org/10.1002/pmic.202000016DOI Listing
July 2020

Bendamustine with total body irradiation conditioning yields tolerant T-cells while preserving T-cell-dependent graft-versus-leukemia.

Oncoimmunology 2020 30;9(1):1758011. Epub 2020 Apr 30.

Department of Pediatrics, University of Arizona, Tucson, AZ, USA.

Graft-versus-host disease (GvHD) remains a significant impediment to allogeneic hematopoietic cell transplantation (HCT) success, necessitating studies focused on alleviating GvHD, while preserving the graft-versus-leukemia (GvL) effect. Based on our previous studies showing bendamustine with total body irradiation (BEN-TBI) conditioning reduces GvHD compared to the current clinical standard of care cyclophosphamide (CY)-TBI in a murine MHC-mismatched bone marrow transplantation (BMT) model, this study aimed to evaluate the role and fate of donor T-cells following BEN-TBI conditioning. We demonstrate that BEN-TBI reduces GvHD compared to CY-TBI independently of T regulatory cells (Tregs). BEN-TBI conditioned mice have a smaller proportion and less activated donor T-cells, with lower CD47 expression, early post-transplant, but no sustained phenotypic differences in T-cells. In BEN-TBI conditioned mice, donor T-cells gain tolerance specific to host MHC antigens. Though these T-cells are tolerant to host antigens, we demonstrate that BEN-TBI preserves a T-cell-dependent GvL effect. These findings indicate that BEN-TBI conditioning reduces GvHD without compromising GvL, warranting its further investigation as a potentially safer and more efficacious clinical alternative to CY-TBI.
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http://dx.doi.org/10.1080/2162402X.2020.1758011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7199810PMC
July 2021
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