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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.

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.

IntroductionMediport use as a clinical option for the administration of chimeric antigen receptor T cell (CAR T cell) therapy in patients with B-cell malignancies has yet to be standardized. Concern for mediport dislodgement, cell infiltration, and ineffective therapy delivery to systemic circulation has resulted in variable practice with intravenous administration of CAR T cell therapy. With CAR T cell commercialization, it is important to establish practice standards for CAR T cell delivery. We conducted a study to establish usage patterns of mediports in the clinical setting and provide a standard of care recommendation for mediport use as an acceptable form of access for CAR T cell infusions. MethodsIn this retrospective cohort study, data on mediport use and infiltration rate was collected from a survey across 34 medical centers in the Pediatric Real-World CAR Consortium, capturing 504 CAR T cell infusion routes across 489 patients. Data represents the largest, and to our knowledge sole, report on clinical CAR T cell infusion practice patterns since FDA approval and CAR T cell commercialization in 2017. ResultsAcross 34 sites, all reported tunneled central venous catheters, including Broviac® and Hickman® catheters, as accepted standard venous options for CAR T cell infusion. Use of mediports as a standard clinical practice was reported in 29 of 34 sites (85%). Of 489 evaluable patients with reported route of CAR T cell infusion, 184 patients were infused using mediports, with no reported incidences of CAR T cell infiltration. Discussion/ConclusionBased on current clinical practice, mediports are a commonly utilized form of access for CAR T cell therapy administration. These findings support the safe practice of mediport usage as an accepted standard line option for CAR T cell infusion.

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

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.

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.

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.

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

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.

Wiskott–Aldrich syndrome (WAS) is an X-linked genetic disorder with a variable phenotypic expression that includes thrombocytopenia, eczema, and immunodeficiency. Some patients may also exhibit autoimmune manifestations. Patients with WAS are at increased risk of developing malignancies such as lymphoma. Allogeneic hematopoietic cell transplantation remains the only curative treatment. Haploidentical bone marrow transplantation (haplo-BMT) with post-transplant cyclophosphamide (PT-CY) has more recently been applied in WAS. Here, we report two brothers who underwent successful T-cell replete haplo-BMT with PT-CY at ages 9 months and 4 years using their father as the donor. Our myeloablative regimen was well-tolerated with minimal organ toxicity and no acute or chronic graft vs. host disease (GvHD). Haplo-BMT may be considered as a safe and effective option for patients with WAS who do not have available human leukocyte antigen (HLA) matched donors.

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.

Primary mediastinal large B-cell lymphoma (PMBCL) is a rare hematologic malignancy with distinct clinical and immunopathological features. We report a case of a young male with disease refractory to multiple lines of therapy, including chimeric antigen receptor-T cells, who achieved his first complete remission after haploidentical bone marrow transplantation (haplo-BMT), following donor leukocyte infusions (DLIs) given concurrently with blinatumomab. While DLI has been used after T-replete haplo-BMT with post-transplant cyclophosphamide, there are no reports on its use for PMBCL. Similarly, blinatumomab is active against B-cell lymphomas, but literature is lacking in patients with PMBCL. Our experience illustrates that blinatumomab can be used concurrently with DLI in a haploidentical setting to achieve disease response in PMBCL. Despite our encouraging experience with this case, we would not recommend this approach outside of a clinical trial as blinatumomab may exacerbate the graft versus host disease risks of DLI, especially in a haploidentical setting. Evaluating this treatment combination in high-risk patients in the setting of a clinical trial may be meaningful.

The treatment of pediatric B-cell acute lymphoblastic leukemia (B-ALL) has undergone several recent advancements, leading to an increased amount of treatment options for relapsed patients. The development of immunotherapies such as anti-CD19 chimeric antigen receptor(CAR) T cells and bispecific T-cell engagers has given clinicians therapeutic options with less expected toxicity when compared to standard re-induction chemotherapy. This is especially beneficial in patients with toxicities from their prior treatment. Along with this, the emergence of haploidentical hematopoietic cell transplantation (HCT) has increased opportunity for patients to receive HCT who may not have had an available matched donor. We present four patients who have received all of these therapies in different combinations to treat multiple relapses. Because of the success of achieving remission as well as decreasing toxicity, the patients are alive and well up to 15 y after the original B-ALL diagnosis, rendering this as a chronic disease for them.

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.

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.

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.

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.

Abstract: Myeloid-derived suppressor cells (MDSCs) are CD11b+Gr1+ cells that induce T-cell hyporesponsiveness, thus impairing antitumor immunity. We have previously reported that disruption of Pak2, a member of the p21-activated kinases (Paks), in hematopoietic stem/progenitor cells (HSPCs) induces myeloid lineage skewing and expansion of CD11bhighGr1high cells in mice. In this study, we confirmed that Pak2-KO CD11bhighGr1high cells suppressed T-cell proliferation, consistent with an MDSC phenotype. Loss of Pak2 function in HSPCs led to (1) increased hematopoietic progenitor cell sensitivity to granulocyte-macrophage colony-stimulating factor (GM-CSF) signaling, (2) increased MDSC proliferation, (3) decreased MDSC sensitivity to both intrinsic and Fas-Fas ligand–mediated apoptosis, and (4) promotion of MDSCs by Pak2-deficient CD4+ T cells that produced more interferon γ, tumor necrosis factor α, and GM-CSF. Pak2 disruption activated STAT5 while downregulating the expression of IRF8, a well-described myeloid transcription factor. Together, our data reveal a previously unrecognized role of Pak2 in regulating MDSC development via both cell-intrinsic and extrinsic mechanisms. Our findings have potential translational implications, as the efficacy of targeting Paks in cancer therapeutics may be undermined by tumor escape from immune control and/or acceleration of tumorigenesis through MDSC expansion.

Objective and Importance. Cyclic neutropenia (CyN) is a rare autosomal dominant inherited disorder due to the mutation ELANE primarily affecting bone marrow stem cells and is characterized by recurrent neutropenia every 2 to 4 weeks. Symptoms vary from benign to severe, including death. Postulations on the cause of wide spectrum in symptom presentation include the possibility of other genetic mutations, such as MEFV. Recommended treatment for CyN is G-CSF to keep ANC higher to minimize risk of infection. Case. A 25-year-old male diagnosed with CyN, on G-CSF but worsening quality of life. Pretransplant investigations revealed ELANE mutation positive severe CyN along with familial Mediterranean fever (MEFV) mutation. Intervention. Bone marrow transplantation as treatment for dual mutation (ELANE and MEFV mutation) positive severe CyN. Conclusion. BMT may be considered as an alternative treatment for severe CyN in patients who are refractory to G-CSF. It is postulated that in our patient the combined mutations (CyN and MEFV) may have contributed to the severity of this individual’s symptoms. We suggest CyN patients who present with severe symptoms have evaluation with ELANE mutation testing, Periodic Fever Syndromes Panel, and routine marrow assessment with FISH, conventional cytogenetics, and morphological evaluation for MDS/AML.

While the role of T helper 17 lymphocytes (Th17) in the pathogenesis of autoimmune diseases and in infectious immunity has been relatively well defined, the impact of these cells and their associated cytokines on cancer development is still under debate. Although multiple reports have indicated that Th17 can promote anticancer immunity, others have argued that these cells may exhibit tumor-promoting properties. This dichotomy in the function of Th17 lymphocytes in cancer may be related to the versatile nature of these cells, being capable of differentiating into either proinflammatory Th1 or suppressive FoxP3-expressing Treg cells or hybrid T cell subsets depending on the underlying environmental conditions. In the current review, we examine the role of Th17 lymphocytes and Th17-associated cytokines in cancer and discuss how factors that control their final lineage commitment decision may influence the balance between their tumor-promoting versus tumor-suppressing properties.

Tumor cells commonly escape from elimination by innate and adaptive immune responses using multiple strategies among which is the active suppression of effector immune cells. Regulatory T lymphocytes (Treg) and tolerogenic dendritic cells play essential roles in the establishment and persistence of cancer-induced immunosuppression. Differentiating dendritic cells (DCs) exposed to tumor-derived factors may be arrested at an immature stage becoming inept at initiating immune responses and may induce effector T-cell anergy or deletion. These tolerogenic DCs, which accumulate in patients with different types of cancers, are also involved in the generation of Treg. In turn, Treg that expand during tumor progression contribute to the immune tolerance of cancer by impeding DCs' ability to orchestrate immune responses and by directly inhibiting antitumoral T lymphocytes. Herein we review these bidirectional communications between DCs and Treg as they relate to the promotion of cancer-induced tolerance.