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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 (pConclusionExercise in humans mobilizes effector lymphocytes with an anti-tumor transcriptomic profile and their use as DLI extends survival and enhances the graft-versus-leukemia (GvL) effect without exacerbating GvHD in human leukemia bearing xenogeneic mice. Exercise may serve as an effective and economical adjuvant to increase the GvL effects of allogeneic cell therapies without intensifying GvHD.

PDF file - 120K, Effects of doxorubicin on the suppressive function of residual MDSC.

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PDF file - 206K, Doxorubicin eliminates tumor-induced MDSC.

PDF file - 124K, Doxorubicin administration increases the effector to suppressor ratios in 4T1 tumor bearing mice but does not alter Treg numbers.

PDF file - 46K, Characterization of in vitro generated Th1 and Th17 lymphocytes.

PDF file - 87K, Doxorubicin is more efficient at selectively eliminating MDSC than cyclophosphamide, fludarabine, melphalan, vinblastine or etoposide.

PDF file - 163K, Effects of doxorubicin on MDSC expression of immunosuppressive factors and on MDSC differentiation.

PDF file - 58K, Doxorubicin eliminates MDSC in mice bearing EMT6 breast cancer or EL4 thymoma.

PDF file - 423K, Doxorubicin increases the frequency, proliferation and cytotoxic activity of effector T lymphocytes and NK.

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.

Allogeneic hematopoietic cell transplantation (allo-HCT) remains a curative option for patients with high-risk myeloid malignancies.We present our 10-year experience (October 2012 to October 2021) of consecutive allo-HCT in patients with myeloid malignancies treated on the pediatric HCT service and conditioned with myeloablative targeted dose-busulfan (BU), fludarabine (FLU), and melphalan (MEL). Twenty-three children, adolescents, and young adult patients (CAYA) (median age 15.4 years) with acute myeloid leukemia (AML, n = 17), myelodysplastic syndrome (MDS, n = 4), or chronic myeloid leukemia (CML, n = 2) underwent allo-HCT post-BU-FLU-MEL. Four patients had treatment-related AML/MDS. Donor/stem cell source was matched sibling donor (MSD) PBSC (n = 7), matched unrelated donor (MUD) PBSC (n = 2), umbilical cord blood (UCB) (n = 3), or haploidentical-BMT (n = 11). Risk stratification was low (n = 2), intermediate (n = 15), high (n = 3), and very high risk (n = 1). The two patients with CML had failed tyrosine kinase inhibitor therapies.With a median follow-up of 41.6 months, the relapse rate is only 4.5% with an overall survival (OS) 100%, progression-free survival (PFS) 95.5%, and graft-versus-host-free-relapse-free survival (GRFS) 67.8%. The donor source and the acute graft-versus-host disease (GvHD) prophylaxis regimen significantly impacted grade II-IV aGvHD 66.7% versus 19.2% (p = .039) and chronic graft-versus-host-disease (cGvHD) 66.7% versus 0% (p = .002) in the patients receiving MSD or MUD PBSC compared to haplo-BMT, respectively, resulting in improved GRFS in haplo-BMT, 83.3% compared to 40% matched donor peripheral blood stem cell transplant (PBSCT) (p = .025).Our results demonstrate that BU-FLU-MEL is efficacious conditioning for disease control in young patients with myeloid malignancies undergoing MSD or alternative donor allo-HCT, but in the setting of PBSC grafts with cyclosporine A-methotrexate (CSA-MTX) GvHD prophylaxis, it results in an unacceptably high incidence of GvHD.

This study aimed to (1) develop TOGETHER-YA, an e-Health-delivered and group-based health-related quality of life (HRQOL) intervention for young adult (YA) cancer survivors aged 18-39 (Part 1), and (2) determine its initial feasibility and acceptability in a single-arm pilot trial (Part 2).TOGETHER-YA is a manualized, 10-week intervention for YA survivors that includes elements of relaxation training, cognitive-behavioral therapy, and health education. In Part 1, content was adapted from existing evidence-based interventions with feedback from YAs (N = 22) in four iterative focus groups. In Part 2, YA survivors (N = 11) participated in a single-arm pilot trial of TOGETHER-YA. Intervention groups were led by a trained facilitator over videoconference. Primary outcomes were feasibility (i.e., recruitment, session attendance, retention) and acceptability (i.e., participant satisfaction).Focus groups reacted positively to TOGETHER-YA and provided actionable recommendations for enhancing its relevance and acceptability, which were implemented. In initial testing, all feasibility and acceptability benchmarks were met; 58% of eligible YAs were recruited, participants attended M = 6 intervention sessions (SD = 3), and 82% of participants were retained post-intervention. On average, participants "agreed" to "strongly agreed" with positive statements about the weekly sessions and the overall program.TOGETHER-YA was developed in collaboration with YA cancer survivors and found to be feasible and acceptable in initial testing. TOGETHER-YA is the first HRQOL intervention for a broad range of YA survivors that is eHealth-delivered for convenience and group-based for peer support. Future large-scale trials should test its efficacy for improving HRQOL.NCT05048316, September 17, 2021; NCT05054569, September 23, 2021.

CD3+/CD56+Natural killer (NK) cell-like T-cells (NKT-like cells) represent ex vivointo cytokine-induced killer (CIK) cells, however this therapeutic cell product has had mixed results against hematological malignancies in clinical trials. The aim of this study was to determine if NKT-like cells mobilized during acute cycling exercise could be used to generate more potent anti-tumor CIK cells from healthy donors. An acute exercise bout increased NKT-like cell numbers in blood 2-fold. Single cell RNA sequencing revealed that exercise mobilized NKT-like cells have an upregulation of genes and transcriptomic programs associated with enhanced anti-tumor activity, including cytotoxicity, cytokine responsiveness, and migration. Exercise, however, did not augment theex vivoexpansion of CIK cells or alter their surface phenotypes after 21-days of culture. CIK cells expanded at rest, during exercise (at 60% and 80% VO2max) or after (1h post) were equally capable of killing leukemia, lymphoma, and multiple myeloma target cells with and without cytokine (IL-2) and antibody (OKT3) primingin vitro. We conclude that acute exercise in healthy donors mobilizes NKT-like cells with an upregulation of transcriptomic programs involved in anti-tumor activity, but does not augment theex vivoexpansion of CIK cells.

The use of immunotherapies has shown promise against selective human cancers. Identifying novel combinations of innate and adaptive immune cell-activating agents that can work synergistically to suppress tumor growth and provide additional protection against resistance or recurrence is critical. The A20 murine lymphoma model was used to evaluate the effect of various combination immunotherapies administered intratumorally. We show that single-modality treatment with Poly(I:C) or GM-CSF-secreting allogeneic cells only modestly controls tumor growth, whereas when given together there is an improved benefit, with 50% of animals clearing tumors and surviving long-term. Neither heat nor irradiation of GM-CSF-secreting cells enhanced the response over use of live cells. The use of a TIM-3 inhibitory antibody and an OX40 agonist in combination with Poly(I:C) allowed for improved tumor control, with 90% of animals clearing tumors with or without a combination of GM-CSF-secreting cells. Across all treatment groups, mice rejecting their primary A20 tumors were immune to subsequent challenge with A20, and this longstanding immunity was T-cell dependent. The results herein support the use of combinations of innate and adaptive immune activating agents for immunotherapy against lymphoma and should be investigated in other cancer types.

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 $$ \dot{\mathrm{V}}{\mathrm{O}}_{2\max } $$ V ̇ 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.

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(2) 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(1 + 2) AR antagonist (nadolol) or a β(1) 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(2) AR mediated, thus highlighting a role for the β(2) 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(2) AR-dependent mobilization and redistribution of VSTs cumulated with each bout of exercise.

Athletes are advised to receive the COVID-19 vaccination to protect them from SARS CoV-2 infection during major competitions. Despite this, many athletes are reluctant to get the COVID-19 vaccine due to concerns that symptoms of vaccinosis may impair athletic performance. OBJECTIVE: To determine the effects of COVID-19 vaccination on the physiological responses to graded exercise. METHODS: Healthy physically active participants completed a 20-minute bout of graded cycling exercise at intensities corresponding to 50, 60, 70 and 80% of the pre-determined VO2max before and ~21 days after receiving the COVID-19 vaccine (2 dose Pfizer mRNA or 1 dose Johnson&Johnson). RESULTS: Vaccination had no effect on a large number of physiological responses to exercise measured in blood (e.g. lactate, epinephrine, cortisol) and by respiratory gas exchange (e.g. oxygen uptake, CO2 production, ventilation, respiratory exchange ratio, predicted VO2max, ventilatory threshold) (p>0.05). We did, however, find significant elevations in heart rate (~5 bpm) and norepinephrine (p = 0.006 and 0.04, respectively) in response to vigorous (e.g. 70-80% VO2max) intensity exercise after vaccination, particularly in those that received the two shot Pfizer mRNA vaccine regimen. These findings held true when compared to demographically matched controls who completed identical bouts of exercise several weeks apart without receiving a vaccine; delta values for heart rate (p=0.03) and norepinephrine (p=0.01) were elevated in the second trial for those that received the Pfizer mRNA vaccine compared to the controls at the 70% and 80% VO2max stages, respectively. CONCLUSION: Recent COVID-19 vaccination has minimal effects on the physiological responses to graded exercise in physically active healthy people. The small elevations in cardiovascular and neuroendocrine responses to exercise after the Pfizer mRNA vaccine regimen could have implications for athletes at the elite level and warrants investigation.

Athletes are advised to receive the COVID-19 vaccination to protect them from SARS CoV-2 infection during major competitions. Despite this, many athletes are reluctant to get the COVID-19 vaccine due to concerns that symptoms of vaccinosis may impair athletic performance.OBJECTIVETo determine the effects of COVID-19 vaccination on the physiological responses to graded exercise.METHODSHealthy participants completed a 20-minute bout of graded cycling exercise before and ~21 days after COVID-19 vaccination (2 dose Pfizer mRNA or 1 dose Johnson & Johnson).RESULTSOxygen uptake, CO2 production, respiratory exchange ratio, ventilation, heart rate, serum noradrenaline, and rating of perceived exertion were significantly elevated in the post vaccine trial. However, vaccination did not affect serum lactate, adrenaline, cortisol, predicted , and ventilatory threshold. Post-vaccine effects on heart rate and noradrenaline remained significant in non-infected participants that received the Pfizer vaccine. No significant effects in respiratory gas exchange parameters were found after vaccination in those previously exposed to SARS-CoV-2, but exercise adrenaline levels were significantly lower and serum lactate levels trending (p= 0.10) lower after vaccination. No changes in any physiological responses to exercise were found in control participants who completed two bouts of exercise separated by ~5 weeks without vaccination.CONCLUSIONRecent COVID-19 vaccination is associated with modest increases in the physiological demands to graded exercise in non-infected healthy people but may actually improve metabolic responses to exercise in those previously infected with SARS-CoV-2. Whether or not these small effects could impact athletic performance at the elite level warrants investigation.

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.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 [Formula: see text] 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.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.

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.

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.

Purpose Total body irradiation (TBI) is an integral part of the conditioning regimen for patients with acute lymphoblastic leukemia (ALL) undergoing allogeneic, hematopoietic, cell transplantation (allo-HCT). There are conflicting data in the literature regarding the utility of a cranial irradiation boost in high-risk adult ALL without evidence of preexisting central nervous system (CNS) involvement. This study investigates the posttransplant clinical outcomes of patients with high-risk adult ALL undergoing TBI conditioning for allo-HCT with or without a whole-brain boost, without overt CNS involvement at the time of diagnosis. Methods and materials A retrospective cohort study was conducted using a medical record analysis. We identified 58 patients who were treated between January 1998 and December 2016, and met our preset inclusion criteria of adults (age >18 years old) who carried a pathologically confirmed diagnosis of CNS-negative, high-risk ALL, who underwent hematopoietic stem cell transplantation with TBI conditioning. A multivariate analysis of correlation between patient outcomes and collected categorical variables was assessed with stepwise Cox logistic regression. Survival analyses were assessed using the Kaplan-Meier technique with a log-rank test. Results With a median follow-up time of 5.3 years, there was a statistically significant improvement in actuarial 7-year CNS relapse-free survival (100% vs 76.4%; P = .043) in favor of patients undergoing a cranial boost. There was no statistically significant improvement in 7-year progression-free survival (78.3% vs 62.5%; P = .076) or overall survival (49.4% vs 43.5%; P = .921) with versus without a cranial boost. On multivariate analysis, the presence of a cranial boost was the only identified variable with an independent relationship to CNS relapse-free survival. Conclusions Adult patients with high-risk, CNS-negative ALL were found to have a statistically significant improvement in CNS relapse-free survival and a trend toward improved progression-free survival with the inclusion of a cranial boost with TBI pretransplant conditioning. Our data indicate that further investigation into the use of cranial boost in this patient population is warranted.

Hepatic late effects are not commonly reported in survivors of childhood leukemia. Four young male patients with acute lymphoblastic leukemia (ALL) were diagnosed with bleeding esophageal varices (EVs) during or shortly after completion of maintenance chemotherapy. EVs were identified from 0 to 60 months after completion of leukemia therapy. All four patients were men between 20 to 24 years old. Hematemesis was the most common presenting symptom. Associated features included splenomegaly, cytopenias, azole therapy, alcohol use, and hepatic iron overload. EVs may be an under-recognized complication of ALL therapy, with adolescent and young adult males at highest risk.

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-IL 15) 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; p10 ±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 γδ-T cells, respectively. This was found to revive the loss in anti-leukemic function observed in vivo when lymphocytes were pre-exposed to SMG. Conclusion: 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.

BACKGROUND: Post-transplant cyclophosphamide (PT-CY) is the most widely applied graft-versus-host disease (GvHD) prophylaxis regimen in T-cell replete haploidentical bone marrow transplantation (haplo-BMT). While PT-CY has been met with great success in the haplo-BMT arena by suppressing GvHD, patients without acute GvHD have high relapse rates. OBJECTIVE: One of the strategies being explored by others to lessen relapse rates is the dose reduction of PT-CY. We have taken a different approach in evaluating whether partially replacing PT-CY with post-transplant bendamustine (PT-BEN) would be advantageous, which is based on our pre-clinical research that delineated several beneficial immunomodulatory properties of BEN. STUDY DESIGN: We therefore initiated and completed a Phase Ia trial which evaluated the progressive substitution of PT-CY with PT-BEN (NCT02996773). Thirteen patients with high-risk hematologic malignancies have received PT-CY/BEN and their outcomes compared to 31 contemporaneous haplo-BMT recipients treated with the same myeloablative conditioning (MAC) regimens but receiving only PT-CY. RESULTS: We demonstrate that partial replacement of PT-CY with PT-BEN on day +4 (PT-CY/BEN) is well tolerated and associated with significantly earlier trilineage engraftment. We also show favorable trends to significant improvements in univariate and multivariate analyses, with PT-CY/BEN compared to PT-CY, with respect to chronic GvHD (HR, 0.08; 95% CI, 0.005, 1.11; P=0.06), and GvHD-free-relapse-free survival (GRFS) (HR, 0.22; 95% CI, 0.05, 0.86; P=0.039). Our human trial has transitioned to Phase Ib which will further evaluate the safety and potential benefits of PT-CY/BEN. Herein, we also expand our pediatric, adolescent, and young adult (AYA) experience to 31 patients demonstrating an overall survival (OS), progression-free survival (PFS) and GRFS at 3 years of 85.6%, 76.1% and 58.2%, respectively in a largely racial/ethnic minority cohort. CONCLUSIONS: PT-CY/BEN appears to be a promising treatment option that requires further evaluation.

Hematopoietic cell transplantation (HCT) is the sole curative option for congenital dyserythropoietic anemia (CDA), a rare type of hemolytic anemia characterized by anemia, ineffective erythropoiesis, and secondary hemochromatosis. In this retrospective multicenter study, we report the outcomes of children with CDA who underwent HCT at participating Pediatric Transplantation and Cellular Therapy Consortium centers. Clinical information on HCT and associated outcomes was collected retrospectively using a common questionnaire. Data were analyzed using descriptive statistics and appropriate analysis. Eighteen patients with CDA who underwent allogeneic HCT between 2002 and 2020 were identified. The majority of patients (n = 13) had CDA type II, and the remainder had either CDA type I (n = 2) or CDA of unknown type (n = 3). Mutations were identified in 7 patients (39%), including SEC23B in 5, GATA1 in 1, and abnormality of chromosome 20 in 1. Thirteen patients had evidence of iron overload pre-HCT and received chelation therapy for a median duration of 10 months (range, 2 months to 17 years) pre-HCT. The median age at the time of HCT was 5.5 years (range, 0.7 to 26 years). Donors were HLA-matched (sibling, 4; unrelated, 10) and mismatched (haploidentical, 1; unrelated, 3). Graft sources were bone marrow in 15 patients, umbilical cord blood in 2 patients, or both in 1 patient. Conditioning included busulfan-based myeloablative (67%), fludarabine-based reduced-intensity (27%), or nonmyeloablative (6%) regimens. Five patients developed veno-occlusive disease, and 4 had viral reactivation. The cumulative incidence of acute graft-versus-host disease (GVHD) was 33%, and that of chronic GVHD was 22%. Four patients (22%) experienced graft failure; all engrafted following either a second HCT (n = 2) or third HCT (n = 2) but sustained considerable morbidities (3 GVHD, 1 death, 2 viral reactivation). With a median follow-up of 3.2 years (range, 0.6 to 14 years)), the 2-year overall survival, event-free survival (EFS), and GVHD-free EFS were 88% (95% confidence interval [CI], 73% to 100%), 65% (95% CI, 45% to 92%), and 60% (95% CI, 40% to 88%), respectively. Univariate analysis did not identify any patient- or transplantation-related variables impacting outcomes. Our study indicates that HCT can be curative for patients with CDA. Strategies such as aggressive chelation, use of preconditioning therapy, and early HCT in the presence of a suitable donor before comorbidities occur are needed to improve engraftment without increasing the risk for toxicity and mortality.

Acute exercise preferentially mobilizes cytotoxic T-cells, NK-cells and non-classical monocytes to the bloodstream under the influence of hemodynamic forces and/or β2-adrenergic receptor (β2-AR) signaling. However, the relative contribution of these mechanisms to the redeployment of the most exercise-responsive cell types is largely unknown. We determined the lymphocyte and monocyte subtypes mobilized to blood during exercise via β2-AR signaling whilst controlling for β1-AR mediated reductions in hemodynamic forces. In a randomized, double blind, complete cross-over design, 14 healthy cyclists exercised for 30-minutes at +10% of blood lactate threshold after ingesting: (1) a placebo, (2) a β1-preferential antagonist (10 mg bisoprolol), or (2) a non-preferential β1 + β2-antagonist (80 mg nadolol) across three trials separated by >7-days. Bisoprolol was administered to reduce hemodynamic forces (heart rate and blood pressure) during exercise to levels comparable with nadolol but without blocking β2-ARs. The mobilization of total NK-cells, terminally differentiated (CD57+) NK-cells, central memory, effector memory and CD45RA+ effector memory CD8+ T-cells; non-classical monocytes; and γδ T-cells were significantly blunted or abrogated under nadolol compared to both bisoprolol and placebo, indicating that the exercise-induced mobilization of these cell types to the blood is largely influenced by β2-AR signaling. Nadolol failed to inhibit the mobilization of classical monocytes, CD4+ T-cells (and their subsets) or naive CD8+ T-cells, indicating that these cell types are mobilized with exercise independently of the β2-AR. We conclude that the preferential mobilization of NK-cells, non-classical monocytes and differentiated subsets of CD8+ T-cells with exercise is largely dependent on catecholamine signaling through the β2-AR. These findings provide mechanistic insights by which distinct lymphocyte and monocyte subtypes are preferentially mobilized to protect the host from anticipated injury or infection in response to an acute stress response.

Haploidentical hematopoietic cell transplant (h-HCT) has become the most frequently applied alternate HCT approach. An obvious benefit of h-HCT is that it offers a potential readily available family donor to almost every patient. The application of h-HCT has particularly benefited minority populations who frequently do not find a suitable unrelated donor. Different options exist utilizing either T cell depletion strategies or T cell-replete grafts with myeloablative or reduced intensity conditioning. Graft failures are generally low (

Twenty-one pediatric and young adult patients (1.1–24.7 years) with hematologic malignancies underwent myeloablative T-cell replete haploidentical bone marrow transplant (haplo-BMT) between October 2015 to December 2019. Fifty-seven percent of the patients were ethnic or racial minorities. Thirteen patients had B-cell precursor acute lymphoblastic leukemia (B-ALL) with 10 receiving 1,200 cGy fractionated total body irradiation with fludarabine while the remaining 11 patients had targeted dose-busulfan, fludarabine, melphalan conditioning. Graft-vs.-host disease (GvHD) prophylaxis consisted of post-transplant cyclophosphamide (15 patients) or cyclophosphamide and bendamustine (six patients), with all patients receiving tacrolimus and mycophenolate mofetil. Twelve patients were in first or second remission at time of transplant with five in >2nd remission and four with measurable disease. Three patients had failed prior transplants and three CAR-T cell therapies. Only one patient developed primary graft failure but engrafted promptly after a second conditioned T-replete peripheral blood stem cell transplant from the same donor. An absolute neutrophil count of 0.5 × 109/L was achieved at a median time of 16 days post-BMT while platelet engraftment occurred at a median of 30 days. The cumulative incidence of grades III to IV acute GvHD and chronic GvHD was 15.2 and 18.1%, respectively. With a median follow-up of 25.1 months the relapse rate is 17.6% with an overall survival of 84.0% and a progression-free survival of 74.3%. The chronic graft-vs.-host-free relapse-free survival (CRFS) is 58.5% while acute and chronic graft-vs.-host-free relapse-free survival (GRFS) is 50.1%. Myeloablative conditioned T-replete haploidentical BMT is a viable alternative to matched unrelated transplantation for children and young adults with high-risk hematologic malignancies.

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/m

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.