Search

Background: The mobilization and redistribution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) specific T-cells and neutralizing antibodies (nAbs) during exercise is purported to increase immune surveillance and protect against severe coronavirus disease 2019 (COVID-19). We sought to determine if COVID-19 vaccination would elicit exercise-responsive SARS-CoV-2 T-cells and transiently alter nAb titers. Methods: Eighteen healthy participants completed a 20-min bout of graded cycling exercise before and/or after receiving a COVID-19 vaccine. All major leukocyte subtypes were enumerated before, during, and after exercise by flow cytometry, and immune responses to SARS-CoV-2 were determined using whole blood peptide stimulation assays, T-cell receptor (TCR)-β sequencing, and SARS-CoV-2 nAb serology. Results: COVID-19 vaccination had no effect on the mobilization or egress of major leukocyte subsets in response to intensity-controlled graded exercise. However, non-infected participants had a significantly reduced mobilization of CD4+ and CD8+ naive T-cells, as well as CD4+ central memory T-cells, after vaccination (synthetic immunity group); this was not seen after vaccination in those with prior SARS-CoV-2 infection (hybrid immunity group). Acute exercise after vaccination robustly mobilized SARS-CoV-2 specific T-cells to blood in an intensity-dependent manner. Both groups mobilized T-cells that reacted to spike protein; however, only the hybrid immunity group mobilized T-cells that reacted to membrane and nucleocapsid antigens. nAbs increased significantly during exercise only in the hybrid immunity group. Conclusion: These data indicate that acute exercise mobilizes SARS-CoV-2 specific T-cells that recognize spike protein and increases the redistribution of nAbs in individuals with hybrid immunity.

Abstract Background The frass of several herbivorous insect species has been utilised as natural medicines in Asia; however, the metabolite makeup and pharmaceutical activities of insect frass have yet to be investigated. Oligophagous Papilionidae insects utilise specific kinds of plants, and it has been suggested that the biochemicals from the plants may be metabolised by cytochrome P450 (CYP) in Papilionidae insects. In this study, we extracted the components of the frass of Papilio machaon larvae reared on Angelica keiskei, Oenanthe javanica or Foeniculum vulgare and examined the biological activity of each component. Then, we explored the expression of CYP genes in the midgut of P. machaon larvae and predicted the characteristics of their metabolic system. Results The components that were extracted using hexane, chloroform or methanol were biochemically different between larval frass and the host plants on which the larvae had fed. Furthermore, a fraction obtained from the chloroform extract from frass of A. keiskei-fed larvae specifically inhibited the cell proliferation of the human colon cancer cell line HCT116, whereas fractions obtained from the chloroform extracts of O. javanica- or F. vulgare-fed larval frass did not affect HCT116 cell viability. The metabolites from the chloroform extract from frass of A. keiskei-fed larvae prevented cell proliferation and induced apoptosis in HCT116 cells. Next, we explored the metabolic enzyme candidates in A. keiskei-fed larvae by RNA-seq analysis. We found that the A. keiskei-fed larval midgut might have different characteristics from the O. javanica- or F. vulgare-fed larval metabolic systems, and we found that the CYP6B2 transcript was highly expressed in the A. keiskei-fed larval midgut. Conclusions These findings indicate that P. machaon metabolites might be useful as pharmaceutical agents against human colon cancer subtypes. Importantly, our findings show that it might be possible to use insect metabolic enzymes for the chemical structural conversion of plant-derived compounds with complex structures.

The most common lymphodepletion regimen used prior to infusion of chimeric antigen receptor-T cells (CAR-T) is cyclophosphamide (CY) in combination with fludarabine (Flu) (CY-FLU). While cyclophosphamide (CY) possesses lymphotoxic effects, it concurrently preserves regulatory T cell activity, potentially affecting the efficacy of CAR-T cells. Moreover, the use of fludarabine (FLU) has been linked to neurotoxicity, which could complicate the early detection of immune effector cell-associated neurotoxicity syndrome (ICANS) observed in CAR-T cell therapy. Given the ongoing shortage of FLU, alternative lymphodepleting agents have become necessary. To date, only a limited number of studies have directly compared different lymphodepleting regimens, and most of these comparisons have been retrospective in nature. Herein, we review the current literature on lymphodepletion preceding CAR-T cell therapies for lymphoid hematologic malignancies, with a specific focus on the use of bendamustine (BEN). Recent evidence suggests that administering BEN before CAR-T cell infusion yields comparable efficacy, possibly with a more favorable toxicity profile when compared to CY-FLU. This warrants further investigation through randomized prospective studies.

BackgroundEvery bout of exercise mobilizes and redistributes large numbers of effector lymphocytes with a cytotoxic and tissue migration phenotype. The frequent redistribution of these cells is purported to increase immune surveillance and play a mechanistic role in reducing cancer risk and slowing tumor progression in physically active cancer survivors. Our aim was to provide the first detailed single cell transcriptomic analysis of exercise-mobilized lymphocytes and test their effectiveness as a donor lymphocyte infusion (DLI) in xenogeneic mice engrafted with human leukemia.MethodsPeripheral blood mononuclear cells (PBMCs) were collected from healthy volunteers at rest and at the end of an acute bout of cycling exercise. Flow cytometry and single-cell RNA sequencing was performed to identify phenotypic and transcriptomic differences between resting and exercise-mobilized cells using a targeted gene expression panel curated for human immunology. PBMCs were injected into the tail vein of xenogeneic NSG-IL-15 mice and subsequently challenged with a luciferase tagged chronic myelogenous leukemia cell line (K562). Tumor growth (bioluminescence) and xenogeneic graft-versus-host disease (GvHD) were monitored bi-weekly for 40-days.ResultsExercise preferentially mobilized NK-cell, CD8+ T-cell and monocyte subtypes with a differentiated and effector phenotype, without significantly mobilizing CD4+ regulatory T-cells. Mobilized effector lymphocytes, particularly effector-memory CD8+ T-cells and NK-cells, displayed differentially expressed genes and enriched gene sets associated with anti-tumor activity, including cytotoxicity, migration/chemotaxis, antigen binding, cytokine responsiveness and alloreactivity (e.g. graft-versus-host/leukemia). Mice receiving exercise-mobilized PBMCs had lower tumor burden and higher overall survival (4.14E+08 photons/s and 47%, respectively) at day 40 compared to mice receiving resting PBMCs (12.1E+08 photons/s and 22%, respectively) from the same donors (p

Abstract Background Immunosenescence is described as age-associated changes within the immune system that are responsible for decreased immunity and increased cancer risk. Physically active individuals have fewer ‘senescent’ and more naïve T-cells compared to their sedentary counterparts, but it is not known if exercise training can rejuvenate ‘older looking’ T-cell profiles. We determined the effects of 12-weeks supervised exercise training on the frequency of T-cell subtypes in peripheral blood and their relationships with circulating levels of the muscle-derived cytokines (i.e. ‘myokines’) IL-6, IL-7, IL-15 and osteonectin in older women at high risk of breast cancer. The intervention involved 3 sessions/week of either high intensity interval exercise (HIIT) or moderate intensity continuous exercise (MICT) and were compared to an untrained control (UC) group. Results HIIT decreased total granulocytes, CD4+ T-cells, CD4+ naïve T-cells, CD4+ recent thymic emigrants (RTE) and the CD4:CD8 ratio after training, whereas MICT increased total lymphocytes and CD8 effector memory (EM) T-cells. The change in total T-cells, CD4+ naïve T-cells, CD4+ central memory (CM) T-cells and CD4+ RTE was elevated after MICT compared to HIIT. Changes in V ̇ O 2 max $$ \dot{\mathrm{V}}{\mathrm{O}}_{2\max } $$ after training, regardless of exercise prescription, was inversely related to the change in highly differentiated CD8+ EMRA T-cells and positively related to changes in β2-adrenergic receptor (β2-AR) expression on CM CD4+ and CM CD8+ T-cells. Plasma myokine levels did not change significantly among the groups after training, but individual changes in IL-7 were positively related to changes in the number of β2-AR expressing CD4 naïve T cells in both exercise groups but not controls. Further, CD4 T-cells and CD4 naive T-cells were negatively related to changes in IL-6 and osteonectin after HIIT but not MICT, whereas CD8 EMRA T-cells were inversely related to changes in IL-15 after MICT but not HIIT. Conclusions Aerobic exercise training alters the frequency of peripheral T-cells associated with immunosenescence in middle aged/older women at high risk of breast cancer, with HIIT (pro-senescent) and MICT (anti-senescent) evoking divergent effects. Identifying the underlying mechanisms and establishing whether exercise-induced changes in peripheral T-cell numbers can alter the risk of developing breast cancer warrants investigation.

Epidemiological data suggest that physical activity protects against severe COVID-19 and improves clinical outcomes, but how exercise augments the SARS-CoV-2 viral immune response has yet to be elucidated. Here we determine the antigen-specific CD4 and CD8 T-cell and humoral immunity to exercise in non-vaccinated individuals with natural immunity to SARS CoV-2, using whole-blood SARS-CoV-2 peptide stimulation assays, IFN-γ ELISPOT assays, 8-color flow cytometry, deep T-cell receptor (TCR) β sequencing, and anti-RBD-1 neutralizing antibody serology. We found that acute exercise reliably mobilized (∼2.5-fold increase) highly functional SARS-CoV-2-specific T-cells to the blood compartment in those with natural immunity to the virus. The mobilized cells reacted with spike protein (including alpha (α) and delta (δ)-variants), membrane, and nucleocapsid peptides in those previously infected but not in controls. Both groups reliably mobilized T-cells reacting with Epstein-Barr viral peptides. Exercise mobilized SARS-CoV-2 specific T-cells maintained broad TCR-β diversity with no impact on CDR3 length or V and J family gene usage. Exercise predominantly mobilized MHC I restricted (i.e. CD8+) SARS-CoV-2 specific T-cells that recognized ORF1ab, surface, ORF7b, nucleocapsid, and membrane proteins. SARS-CoV-2 neutralizing antibodies were transiently elevated ∼1.5-fold during exercise after infection. In conclusion, we provide novel data on a potential mechanism by which exercise could increase SARS-CoV-2 immunosurveillance via the mobilization and redistribution of antigen-specific CD8 T-cells and neutralizing antibodies. Further research is needed to define the tissue specific disease protective effects of exercise as SARS-CoV-2 continues to evolve, as well as the impact of COVID-19 vaccination on this response.

IntroductionThe use of allogeneic hematopoietic cell transplantation (allo-HCT) for treating hematological disorders is increasing, but the development of graft-versus-host disease (GvHD) remains a major cause of morbidity and mortality. The use of post-transplant cyclophosphamide (CY) has significantly improved outcomes following allo-HCT, but complications of viral reactivation due to delayed immune reconstitution or relapse remain. Other laboratories are evaluating the potential benefit of lowering the dose of CY given post-transplant, whereas our laboratory has been focusing on whether partially replacing CY with another DNA alkylating agent, bendamustine (BEN) may be advantageous in improving outcomes with allo-HCT.MethodsHere, we utilized a xenogeneic GvHD (xGvHD) model in which immunodeficient NSG mice are infused with human peripheral blood mononuclear cells (PBMCs).ResultsWe show that a lower dose of CY (25 mg/kg) given on days +3 and +4 or CY (75 mg/kg) given on only day +3 post-PBMC infusion is not sufficient for improving survival from xGvHD, but can be improved with the addition of BEN (15 mg/kg) on day +4 to day +3 CY (75 mg/kg). CY/BEN treated mice when combined with cyclosporine A (CSA) (10mg/kg daily from days +5 to +18 and thrice weekly thereafter), had improved outcomes over CY/CY +CSA treated mice. Infiltration of GvHD target organs was reduced in both CY/CY and CY/BEN treatment groups versus those receiving no treatment. CY/CY +CSA mice exhibited more severe xGvHD at day 10, marked by decreased serum albumin and increased intestinal permeability. CY/BEN treated mice had reductions in naïve, effector memory and Th17 polarized T cells. RNAseq analysis of splenocytes isolated from CY/CY and CY/BEN treated animals revealed increased gene set enrichment in multiple KEGG pathways related to cell migration, proliferation/differentiation, and inflammatory pathways, among others with CY/BEN treatment.ConclusionTogether, we illustrate that the use of CY/BEN is safe and shows similar control of xGvHD to CY/CY, but when combined with CSA, survival with CY/BEN is significantly prolonged compared to CY/CY.

Abstract 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.

CD3+/CD56+ Natural killer (NK) cell-like T-cells (NKT-like cells) represent

Rai et al. characterise the properties of secreted midbody remnants, showing they are distinct from exosomes and microvesicles. The authors also find that these vesicles are engulfed by cells and promote anchorage independent growth and invasive phenotypes in NIH3T3 fibroblasts.

Abstract We have initiated a single center phase I study in patients with hematologic malignancies progressively substituting day +4 posttransplant cyclophosphamide (PT‐CY) with bendamustine (PT‐BEN) following myeloablative conditioning (MAC) and T‐cell replete haploidentical bone marrow transplantation (haplo‐BMT). We report herein our interim analysis of our first three cohorts PT‐CY (mg/kg)/PT‐BEN (mg/m2): 40/20, 20/60, and 0/90. All patients have tolerated PT‐CY/BEN well with no dose limiting toxicities. Compared to contemporaneous controls undergoing haplo‐BMT with the same MAC regimens but only PT‐CY, we have observed earlier trilineage engraftment (P = .002 neutrophils, P = .014 platelets) and a lower incidence of cytomegalovirus reactivation (P = .016) in the PT‐CY/BEN cohorts. After substituting day +4 PT‐CY with PT‐BEN, the registered trial (www.clinicaltrials.gov; NCT02996773) is proceeding to replace day +3 PT‐CY with PT‐BEN with a view to identifying further evidence on the potential advantages of PT‐BEN.

The immediate precursor to murine type 1 conventional DCs (cDC1s) has recently been established and named "pre-cDC1s". Mature CD8α+ cDC1s are recognized for suppressing graft-versus-host disease (GvHD) while promoting graft-versus-leukemia (GvL), however pre-cDC1s have not previously been investigated in the context of alloreactivity or anti-tumor responses. Characterization of pre-cDC1s, compared to CD8α+ cDC1s, found that a lower percentage of pre-cDC1s express PD-L1, yet express greater PD-L1 by MFI and a greater percent PIR-B, a GvHD-suppressing molecule. Functional assays were performed ex vivo following in vivo depletion of CD8α+ DCs to examine whether pre-cDC1s play a redundant role in alloreactivity. Proliferation assays revealed less allogeneic T-cell proliferation in the absence of CD8α+ cDC1s, with slightly greater CD8+ T-cell proliferation. Further, in the absence of CD8α+ cDC1s, stimulated CD8+ T-cells exhibited significantly less PD-1 expression compared to CD4+ T-cells, and alloreactive T-cell death was significantly lower, driven by reduced CD4+ T-cell death. Tumor-killing assays revealed that T-cells primed with CD8α-depleted DCs ex vivo induce greater killing of A20 B-cell leukemia cells, particularly when antigen (Ag) is limited. Bulk RNA sequencing revealed distinct transcriptional programs of these DCs, with pre-cDC1s exhibiting activated PD-1/PD-L1 signaling compared to CD8α+ cDC1s. These results indicate distinct T-cell-priming capabilities of murine pre-cDC1s compared to CD8α+ cDC1s ex vivo, with potentially clinically relevant implications in suppressing GvHD while promoting GvL responses, highlighting the need for greater investigation of murine pre-cDC1s.

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, ex vivo 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 ex vivo peptide stimulation and maintained broad TCR-β diversity against SARS-CoV-2 antigens both before and after ex vivo 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.

Abstract 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.

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 in-vivo 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.

IntroductionOlder 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.PurposeCompare 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.MethodsTwenty 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.ResultsNo significant group x time effects were found in antibody responses to any strain (interaction for A/H1N1: p = 0.682; A/H3N2: p = 0.644; B/Colorado/06/2017: p = 0.262; B/Phuket/3073/2013: p = 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: p = 0.985; 6-weeks: p = 0.889; 24 weeks: p = 0.857). One subject (Ex-S) reported flu-like symptoms 18 weeks post-vaccination.ConclusionAcute 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.

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.

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 via 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.

Background: We have previously shown that the anti-tumor activity of human lymphocytes is diminished in vitro after 12-hours pre-exposure to simulated microgravity (SMG). Here we used an immunocompromised mouse model to determine if this loss of function would extend in vivo, and to also test the efficacy of IL-2 and zoledronic acid (ZOL) therapy as a potential countermeasure against SMG-induced immune dysfunction. We adoptively transferred human lymphocytes that were exposed to either SMG or 1G-control into NSG-Tg (Hu-IL15) mice 1-week after they were injected with a luciferase-tagged human chronic myeloid leukemia (K562) cell line. Tumor growth was monitored 2x weekly with bioluminescence imaging (BLI) for up to 6-weeks. Results: Mice that received lymphocytes exposed to SMG showed greater tumor burden compared to those receiving lymphocytes exposed to 1G (week 6 BLI: 1.8e10 ± 8.07e9 versus 2.22e8 ± 1.39e8 photons/second; p < 0.0001). Peak BLI was also higher in the SMG group compared to 1G-control (2.34e10 ± 1.23e10 versus 3.75e8 ± 1.56e8 photons/second; p = 0.0062). Exposure to SMG did not affect the ability of human lymphocytes to engraft or evoke xeno-graft-versus-host disease in the mice. Additionally, we injected the mice with IL-2 and zoledronic acid (ZOL) to expand and activate the anti-tumor activity of NK cells and γ δ-T cells, respectively. This treatment was found to revive the loss of anti-leukemic function observed in vivo when lymphocytes were pre-exposed to SMG. Conclusions: Microgravity plays a contributory role in loss of tumor control in vivo. Immuno-stimulating agents like ZOL+IL-2 may offer an important countermeasure for immune dysregulation during prolonged spaceflight.

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.

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

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.

Abstract Background Transformation by oncogene Ras overcomes TGF-β mediated growth inhibition in epithelial cells. However, it cooperates with each other to mediate epithelial to mesenchymal transition (EMT). The mechanism of how these two pathways interact with each other is controversial. Methods Molecular techniques were used to engineer expression plasmids for Ras, SPRY, TGF-β receptors, type I and II and ubiquitin. Immunoprecipitation and western blots were employed to determine protein-protein interactions, preotein levels, protein phosphorylation while immunofluorecesent staining for molecular co-localization. TGF-β signalling activities is also determined by its luciferase reporter assay. Trans-well assays were used to measure cell migration and invasion. Results Ras interacts with the SPSB1’s SPRY domain to enhance TGF-β signaling. Ras interacts and colocalizes with the TGF-β type II receptor’s (TβRII) negative regulator SPSB1 on the cell membrane, consequently promoting SPSB1 protein degradation via enhanced mono- and di-ubiquitination. Reduced SPSB1 levels result in the stablization of TβRII, in turn the increase of receptor levels significantly enhance Smad2/3 phosphorylation and signaling. Importantly, forced expression of SPSB1 in Ras transformed cells suppresses TGF-β signaling and its mediated migration and invasion. Conclusion Ras positively cooperates with TGF-β signaling by reducing the cellular protein levels of TβRII negative regualtor SPSB1.

Abstract The 43-kDa trans-activating response region DNA-binding protein 43 (TDP-43) is a product of a causative gene for amyotrophic lateral sclerosis (ALS). Despite of accumulating evidence that mitochondrial dysfunction underlies the pathogenesis of TDP-43–related ALS, the roles of wild-type TDP-43 in mitochondria are unknown. Here, we show that the small TDP-43 population present in mitochondria binds directly to a subset of mitochondrial tRNAs and precursor RNA encoded in L-strand mtDNA. Upregulated expression of TDP-43 stabilised the processing intermediates of mitochondrial polycistronic transcripts and their products including the components of electron transport and 16S mt-rRNA, similar to the phenotype observed in cells deficient for mitochondrial RNase P. Conversely, TDP-43 deficiency reduced the population of processing intermediates and impaired mitochondrial function. We propose that TDP-43 has a novel role in maintaining mitochondrial homeostasis by regulating the processing of mitochondrial transcripts.

TCR-gamma delta (γδ) T-cells are considered important players in the graft-vs.-tumor effect following allogeneic hematopoietic cell transplantation (alloHCT) and have emerged as candidates for adoptive transfer immunotherapy in the treatment of both solid and hematological tumors. Systemic β-adrenergic receptor (β-AR) activation has been shown to mobilize TCR-γδ T-cells to the blood, potentially serving as an adjuvant for alloHCT and TCR-γδ T-cell therapy. We investigated if systemic β-AR activation, using acute dynamic exercise as an experimental model, can increase the mobilization, ex vivo expansion, and anti-tumor activity of TCR-γδ T-cells isolated from the blood of healthy humans. We also sought to investigate the β-AR subtypes involved, by administering a preferential β1-AR antagonist (bisoprolol) and a non-preferential β1 + β2-AR antagonist (nadolol) prior to exercise as part of a randomized placebo controlled cross-over experiment. We found that exercise mobilized TCR-γδ cells to blood and augmented their ex vivo expansion by ~182% compared to resting blood when stimulated with IL-2 and ZOL for 14-days. Exercise also increased the proportion of CD56+, NKG2D+/CD62L–, CD158a/b/e+ and NKG2A− cells among the expanded TCR-γδ cells, and increased their cytotoxic activity against several tumor target cells (K562, U266, 221.AEH) in vitro by 40–60%. Blocking NKG2D on TCR-γδ cells in vitro eliminated the augmented cytotoxic effects of exercise against U266 target cells. Furthermore, administering a β1 + β2-AR (nadolol), but not a β1-AR (bisoprolol) antagonist prior to exercise abrogated the exercise-induced enhancement in TCR-γδ T-cell mobilization and ex vivo expansion. Furthermore, nadolol completely abrogated while bisoprolol partially inhibited the exercise-induced increase in the cytotoxic activity of the expanded TCR-γδ T-cells. We conclude that acute systemic β-AR activation in healthy donors markedly augments the mobilization, ex vivo expansion, and anti-tumor activity of TCR-γδ T-cells and that some of these effects are due to β2-AR signaling and phenotypic shifts that promote a dominant activating signal via NKG2D. These findings highlight β-ARs as potential targets to favorably alter the composition of allogeneic peripheral blood stem cell grafts and improve the potency of TCR-γδ T-cell immune cell therapeutics.

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.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Extracellular vesicle (EV) microRNAs are of major interest as potential diagnostic biomarkers in all cancer types. This study aims to identify miRNA profiles of shed microvesicles (sMVs) and exosomes (Exos) secreted from the isogenic colorectal cancer (CRC) cell lines SW480 and SW620 and evaluate their ability to predict CRC. Deep sequencing of miRNAs in parental cell lysates (CLs) and highly-purified sMVs and Exos was performed. We focused on miRNAs enriched in EVs and dysregulated miRNAs in metastatic cells (SW620) relative to primary cancer cells (SW480). We investigated the ability of EV miRNA signatures to predict CRC tumours using 594 tumours (representing different pathological stages) and 11 normal samples obtained from TCGA. In SW480 and SW620 cells we identified 345 miRNAs, of which 61 and 73 were upregulated and downregulated in SW620-CLs compared to SW480-CLs, respectively. Selective distribution of cellular miRNAs into EVs results in distinct miRNA signatures for sMVs and Exos in each cell line. Cross cell line comparisons of EV miRNA profiles reveal a subset of miRNAs critical in CRC progression from primary carcinoma to metastasis. Many miRNAs non-detectable (1000 TPM) in secreted EVs. Strikingly, miR-7641 which is non-detectable in SW480-CL but upregulated in SW620-CL is highly enriched in EVs secreted from both cell lines. Pearson correlation analysis demonstrated that EV miRNA profiles can be used to predict CRC tumours with ~96% accuracy. Our findings suggest that EV miRNA profiles from CRC cell lines may allow prediction of CRC tumours, and that miR-7641 may serve as an attractive candidate for the specific, non-invasive diagnosis and prognosis of CRC.

Recent studies have established that dysregulation of the human immune system and the reactivation of latent herpesviruses persists for the duration of a 6-month orbital spaceflight. It appears certain aspects of adaptive immunity are dysregulated during flight, yet some aspects of innate immunity are heightened. Interaction between adaptive and innate immunity also seems to be altered. Some crews experience persistent hypersensitivity reactions during flight. This phenomenon may, in synergy with extended duration and galactic radiation exposure, increase specific crew clinical risks during deep space exploration missions. The clinical challenge is based upon both the frequency of these phenomena in multiple crewmembers during low earth orbit missions and the inability to predict which specific individual crewmembers will experience these changes. Thus, a general countermeasure approach that offers the broadest possible coverage is needed. The vehicles, architecture, and mission profiles to enable such voyages are now under development. These include deployment and use of a cis-Lunar station (mid 2020s) with possible Moon surface operations, to be followed by multiple Mars flyby missions, and eventual human Mars surface exploration. Current ISS studies will continue to characterize physiological dysregulation associated with prolonged orbital spaceflight. However, sufficient information exists to begin consideration of both the need for, and nature of, specific immune countermeasures to ensure astronaut health. This article will review relevant in-place operational countermeasures onboard ISS and discuss a myriad of potential immune countermeasures for exploration missions. Discussion points include nutritional supplementation and functional foods, exercise and immunity, pharmacological options, the relationship between bone and immune countermeasures, and vaccination to mitigate herpes (and possibly other) virus risks. As the immune system has sentinel connectivity within every other physiological system, translational effects must be considered for all potential immune countermeasures. Finally, we shall discuss immune countermeasures in the context of their individualized implementation or precision medicine, based on crewmember specific immunological biases.