Aims: Vitamin C (ascorbic acid) is thought to enhance immune function, but the mechanisms involved are obscure. We utilized an in vitro model of T-cell maturation to evaluate the role of ascorbic acid in lymphocyte development. Results: Ascorbic acid was essential for the developmental progression of mouse bone marrowderived progenitor cells to functional T-lymphocytes in vitro and also played a role in vivo. Ascorbate-mediated enhancement of T-cell development was lymphoid cell-intrinsic and independent of T-cell receptor (TCR) rearrangement. Analysis of TCR rearrangements demonstrated that ascorbic acid enhanced the selection of functional TCRab after the stage of b-selection. Genes encoding the coreceptor CD8 as well as the kinase ZAP70 were upregulated by ascorbic acid. Pharmacologic inhibition of methylation marks on DNA and histones enhanced ascorbate-mediated differentiation, suggesting an epigenetic mechanism of Cd8 gene regulation via active demethylation by ascorbate-dependent Fe 2+ and 2-oxoglutarate-dependent dioxygenases. Innovation: We speculate that one aspect of gene regulation mediated by ascorbate occurs at the level of chromatin demethylation, mediated by Jumonji C ( JmjC) domain enzymes that are known to be reliant upon ascorbate as a cofactor. JmjC domain enzymes are also known to regulate transcription factor activity. These two mechanisms are likely to play key roles in the modulation of immune development and function by ascorbic acid. Conclusion: Our results provide strong experimental evidence supporting a role for ascorbic acid in T-cell maturation as well as insight into the mechanism of ascorbate-mediated enhancement of immune function.

The association between pre-hematopoietic stem cell transplantation (HSCT) vancomycin-resistant Enterococcus (VRE) colonization, HSCT-associated VRE bacteremia, and HSCT mortality is disputed. We studied 161 consecutive patients with acute leukemia who underwent HSCT at our hospital between 2006 and 2014, of whom 109 also received leukemia induction/consolidation on our unit. All inpatients had weekly VRE stool surveillance. Pre-HSCT colonization was not associated with increases in HSCT mortality but did identify a subgroup of HSCT recipients with a higher risk for VRE bacteremia and possibly bacteremia from other organisms. The major risk factor for pre-HSCT colonization was the number of hospital inpatient days between initial admission for leukemia and HSCT. One-third of evaluable patients colonized before HSCT were VRE-culture negative on admission for HSCT; these patients had an increased risk for subsequent VRE stool surveillance positivity but not VRE bacteremia. Molecular typing of VRE isolates obtained before and after HSCT showed that VRE strains frequently change. Postengraftment VRE bacteremia was associated with a much higher mortality than pre-engraftment VRE bacteremia. Pre-engraftment bacteremia from any organism was associated with an alternative donor and resulted in an increase in hospital length of stay and cost. Mortality was similar for pre-engraftment VRE bacteremia and pre-engraftment bacteremia due to other organisms, but mortality associated with post-engraftment VRE bacteremia was higher and largely explained by associated severe graft-versus-host disease and relapsed leukemia. These data emphasize the importance of distinguishing between VRE colonization before HSCT and at HSCT, between pre-engraftment and postengraftment VRE bacteremia, and between VRE bacteremia and bacteremia from other organisms.

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm caused by BCR-ABL, a constitutively active tyrosine kinase generated as a result of the t(9;22)(q34;q11). The natural history of CML is progression from a relatively benign chronic phase to an acute leukemia termed blast crisis. Imatinib, an inhibitor of BCR-ABL tyrosine kinase activity, has a dramatic effect on the natural history of the disease. Despite the favorable outcomes with imatinib, a subset of patients have primary refractory disease, or experience relapse after an initial response. Recently identified molecular predictors of drug response might help predict outcome with tyrosine kinase inhibitor therapy more accurately than clinical prognostication scores, but have not yet been introduced into clinical routine. These techniques include analysis of drug transport proteins, in vitro drug assays, measurement of imatinib plasma levels, BCR-ABL activity monitoring, and gene expression profiling. In this article we review the current status of these technologies, which may ultimately allow us to tailor therapy to a specific patient.

2623 Introduction: A complete understanding of factors that drive T cell differentiation from hematopoietic progenitors remains a fundamental goal of hematology. T cells are key regulators and effectors in defense against infections and malignancies. Aberrations in T cell regulation play causative roles in autoimmune and graft-versus-host disease. The OP9-DL1 stromal cell line enables investigation of lymphocyte development in vitro. Lymphocyte progenitors (KLS, Thy1.1 -) harvested from murine adult bone marrow and seeded onto the OP9-DL1 stromal line can be followed through stages of maturation by immunophenotyping. We previously demonstrated that addition of a stabilized form of vitamin C, phospho-ascobate (pAsc), to culture media promoted T lineage differentiation. To address the mechanism of this effect, we employed quantitative RT-PCR and spectratyping analysis to examine mRNA expression of rearranged complementarity-determining region 3 (CDR3) polymorphisms in T cell receptor (TCR) beta variable (BV) and alpha variable (AV) genes in the presence or absence of pASC. Methods: Lymphocyte progenitor cells (KLS, Thy1.1-) were sorted from adult mouse bone marrow and 1000–2000 progenitors were seeded per well in a 24 well plate coated with OP9-DL1 stromal cells. Cultures were supplemented with IL-7 (5 ng/ml), Flt3 ligand (5 ng/mL) and SCF (100 ng/mL) plus or minus pAsc (100 mcg/mL). Cells were passaged, counted and reseeded with fresh media and supplements twice a week over a 21 day period. Immunophenotype and viability were evaluated by flow cytometry. Markers for T cell development included CD44, CD25, CD3, CD4, CD8, TCR beta chain and TCR gamma-delta chains. Total RNA from cultured cells was isolated at day 21, reverse transcribed to cDNA, and analyzed by RT-PCR for differential expression of BV and AV genes using gene-specific primers for BV1, BV4, BV8.2, BV13, AV2, and AV8 with corresponding beta constant (BC) and alpha constant (AC) primers. For spectratyping, RT-PCR amplicons were generated using BV or AV gene-specific primers for BV1, BV4, BV8.2, BV13, AV1, AV2, AV5, AV8, AV10, AV13, AV16, AV18, and AV19 with corresponding BC and AC primers. These products were then re-amplified with the same gene-specific primers but with fluorochrome-labeled nested BC or AC primers. Spectratype analysis was performed on labeled amplicons by capillary electrophoresis. Results: T cell differentiation was markedly advanced by the addition of pAsc, with the majority of cells co-expressing CD4/CD8 and TCR beta/CD3. Transfection of a functionally rearranged TCR beta gene failed to rescue cells cultured without pAsc to the double positive stage; similar results were obtained with bone marrow cells derived from TCR alpha-beta transgenic donor mice. Cells cultured with pAsc demonstrated an average 5 fold increase (5.08 ± 0.40) in expression of BV genes and an average 13 fold increase (13.46 ± 2.18) of AV genes. pAsc did not induce alterations in the spectratype distributions of BV amplicons compared those generated under non-pAsc conditions, nor to distributions derived from thymic cDNA. However, spectratype distributions of AV amplicons generated under pAsc conditions more closely resembled those derived from thymic and lymph node cDNA than distributions generated from non-pAsc conditions. Conclusions: In our in vitro model, the addition of pAsc promotes robust differentiation of adult mouse bone marrow progenitors to T cells co-expressing CD4/CD8 and alpha-beta TCR. However, the mechanism by which pAsc exerts its effect remains elusive. We suspect that pAsc enhances an already pre-programmed process. The fact that transfection of a functional TCR beta gene fails to rescue differentiation, coupled with our observation that pAsc has no effect on BV spectratypes suggests that enhancement of beta selection is not involved. Rather, the AV spectratyping data suggest that pAsc exerts its effect temporally near alpha gene rearrangement, possibly via enhancement of the TCR signal transduction cascade. Further work will include PCR microarray analysis encompassing multiple signal transduction cascades involved in hematopoietic progenitor differentiation. These findings will help develop a model for future mechanistic studies and for ex vivo expansion of immune cells for therapeutic use. Disclosures: No relevant conflicts of interest to declare.