Key Points• Number of prior chemotherapy cycles in cancer patients correlates with T-cell senescent phenotype and loss of CD27 and CD28 expression.• Addition of PI3Kd inhibitors and VIP antagonists increased ex vivo expansion, in vivo persistence, and anticancer cytotoxicity of T cells.

Key Points• Hb-conformation-dependent interaction with band 3 protein regulates glycolysis in RBCs.• In hypoxia, HbS disrupts this system, disabling RBC antioxidant defense. Energy metabolism in RBCs is characterized by O 2 -responsive variations in flux through the Embden Meyerhof pathway (EMP) or the hexose monophosphate pathway (HMP). Therefore, the generation of ATP, NADH, and 2,3-DPG (EMP) or NADPH (HMP) shift with RBC O 2 content because of competition between deoxyhemoglobin and key EMP enzymes for binding to the cytoplasmic domain of the Band 3 membrane protein (cdB3). Enzyme inactivation by cdB3 sequestration in oxygenated RBCs favors HMP flux and NADPH generation (maximizing glutathione-based antioxidant systems). We tested the hypothesis that sickle hemoglobin disrupts cdB3-based regulatory protein complex assembly, creating vulnerability to oxidative stress. In RBCs from patients with sickle cell anemia, we demonstrate in the present study constrained HMP flux, NADPH, and glutathione recycling and reduced resilience to oxidative stress manifested by membrane protein oxidation and membrane fragility. Using a novel, inverted membrane-on-bead model, we illustrate abnormal (O 2 -dependent) association of sickle hemoglobin to RBC membrane that interferes with sequestration/inactivation of the EMP enzyme GAPDH. This finding was confirmed by immunofluorescent imaging during RBC O IntroductionSickle cell anemia (SCA) arises from a single amino acid substitution (Glu6Val) in the ␤-globin chain. Although the change to hemoglobin (Hb) is simple and uniform, SCA is characterized by broad differences in clinical manifestation. Phenotype variation in SCA is thought to arise from both environmental and genetic factors (eg, ␤-gene cluster haplotype, degree of HbF expression, or effects of other epistatic genes). The environmental factor that most clearly influences SCA phenotype is hypoxia, which drives sickle Hb (HbS) polymerization and the resulting well-characterized alterations in RBC physiology and the microcirculation. However, the influence of hypoxia on the SCA phenotype appears to be insufficiently explained by HbS polymerization alone. 1 Moreover, we lack a clear mechanistic understanding of the significant oxidative stress complicating SCA, a key feature of phenotype variation, both at rest and in association with hypoxia. 2 Nonpolymerized, solution-phase HbS may promote oxidative stress, even in RBCs under normal physiologic O 2 gradients. 3 Specifically, the low redox potential for heme in HbS 4 and avid binding affinity of HbS for the cytoplasmic regulatory domain of the Band 3 membrane protein (cdB3) 5,6 strongly affect RBC energetics and antioxidant systems [7][8][9] and, notably, do so as a function of RBC O 2 content. Therefore, both the genesis and the disposal of reactive oxygen species are abnormal in SCA, creating a baseline state of oxidative stress, which worsens in hypoxia.In particular, consideration of metabolic control in RBCs suggests O 2 -dependent HbS-cdB3 interaction as a relatively ...

A higher number of donor plasmacytoid dendritic cells (pDCs) is associated with increased survival and reduced graft-versus-host disease (GVHD) in human recipients of unrelated donor bone marrow (BM) grafts, but not granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood grafts. We show that in murine models, donor BM pDCs are associated with increased survival and decreased GVHD compared with G-CSF-mobilized pDCs. To increase the content of pDCs in BM grafts, we studied the effect of FMS-like tyrosine kinase 3 ligand (Flt3L) treatment of murine BM donors on transplantation outcomes. Flt3L treatment (300 mg/kg/day) resulted in a scheduledependent increase in the content of pDCs in the BM. Mice treated on days -4 and -1 had a >5-fold increase in pDC content without significant changes in numbers of HSCs, T cells, B cells, and natural killer cells in the BM graft. In an MHC-mismatched murine transplant model, recipients of Flt3L-treated T cell-depleted (TCD) BM (TCD F-BM) and cytokine-untreated T cells had increased survival and decreased GVHD scores with fewer Th1 and Th17 polarized T cells post-transplantation compared with recipients of equivalent numbers of untreated donor TCD BM and T cells. Gene array analyses of pDCs from Flt3L-treated human and murine donors showed up-regulation of adaptive immune pathways and immunoregulatory checkpoints compared with pDCs from untreated BM donors. Transplantation of TCD F-BM plus T cells resulted in no loss of the graft-versus-leukemia (GVL) effect compared with grafts from untreated donors in 2 murine GVL models. Thus, Flt3L treatment of BM donors is a novel method for increasing the pDC content in allografts, improving survival, and decreasing GVHD without diminishing the GVL effect.

Vasoactive intestinal peptide (VIP), an anti-inflammatory neuropeptide with pleiotropic cardio-vascular effects, induces differentiation of hematopoietic stem cells into regulatory dendritic cells that limit graft-versus-host disease (GvHD) in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We have previously shown that donor plasmacytoid dendritic cells (pDCs) in bone marrow (BM) donor grafts limit the pathogenesis of GvHD. In this current study we show that murine and human pDCs express VIP, and that VIP-expressing pDCs limit T cell activation and expansion using both in vivo and in vitro model systems. Using T cells or pDCs from transgenic luciferase+ donors in murine BMT, we show similar homing patterns of donor pDCs and T cells to the major sites for allo-activation of donor T cells: spleen, and gut. Co-transplanting VIP-KO pDCs with hematopoietic stem cells (HSCs) and T cells in MHC mis-matched allogeneic BMT led to lower survival, higher GvHD scores, and more colon crypt cell apoptosis than transplanting wild-type pDCs. BMT recipients of VIP-KO pDCs had more Th1 polarized T cells, and higher plasma levels of GM-CSF and TNF-alpha than recipients of wild-type pDCs. T cells from VIP-KO pDC recipients had increasing levels of bhlhe40 transcripts during the first two weeks post-transplant, and higher levels of CyclophilinA/Ppia transcripts at day 15 compared with T cells from recipients of wild type pDCs. Collectively, these data indicate paracrine VIP synthesis by donor pDCs limits pathogenic T cell inflammation, supporting a novel mechanism by which donor immune cells regulate T cell activation and GvHD in allogeneic BMT.