Bone marrow preconditioning using cyclophosphamide (CP) is generally used for bone marrow transplantation (BMT). However, because of CP's hepatotoxicity and nephrotoxicity, additional fludarabine (FDR) administration and a reduced dose of CP are used for reduced-intensity preconditioning. Recently, preclinical studies using non-human primates (NHPs) were performed to induce immune tolerance after solid organ transplantation by conducting BMT simultaneously. However, dose optimization of CP and FDR for BMT preconditioning in cynomolgus monkeys has not been conducted. Therefore, the objective of this study was to evaluate the efficacy and tolerability of induction protocols using different doses of CP and FDR. Our results showed that relatively low-dose CP (30 mg/kg×2) combined with additional high-dose FDR (60 mg/m2×4) was associated with sufficient suppression in periphery as well as in bone marrow compared with high-dose CP (60 mg/kg×2) combined with low-dose FDR (30 mg/m2×4) and did not show hepatic or renal toxicity. CD34+ stem cells were also well suppressed with both doses. Therefore, we concluded that the combination of 60 mg/kg of CP with 240 mg/m2 of FDR can be used effectively and safely for non-myeloablative preconditioning for BMT in cynomolgus monkeys.
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Several lines of evidence support the concept that NK cells play an important role in control of hepatitis C virus (HCV) infection via cytokine secretion and cytotoxicity. IL-7 is a homeostatic cytokine with a role in T cell development, activation, proliferation, and cytokine secretion. The IL-7Rα chain [cluster of differentiation (CD)127] is expressed on NK cells, with greatest abundance on the CD56brightCD16dim/- (CD56bright) subset. Here, we measured CD127 expression on CD56bright, CD56dimCD16+ (CD56dim), or CD56negCD16+ (CD56neg) NK cell subsets of 25 uninfected donors (UD); 34 chronic HCV-infected, treatment-naïve; 25 HIV-infected, virally suppressed on antiretroviral therapy (ART); and 42 HCV-HIV-coinfected subjects on ART. Interestingly, CD127 expression on CD56bright NK cells negatively correlated with HCV plasma levels in HCV monoinfection and HCV-HIV coinfection. IL-7 induced CD69 expression, as well as IFN-γ production, in CD56bright NK cells and also enhanced the IFN-α-induced CD69 expression on these cells. The latter was impaired in HIV infection. Furthermore, IL-7 induced B cell lymphoma 2 (BCL-2) expression and cell cycling of CD56bright NK cells, and this effect was impaired in HCV- and HIV-infected subjects. Whereas IL-7-stimulated CD56bright NK cell degranulation appeared intact in all cohorts, we observed impaired IL-7-activated NK cell cytolytic function in HCV- and HIV-infected subjects. Finally, IL-7-induced phosphorylation of STAT-5 (pSTAT-5) signaling was impaired in NK cells of subjects with chronic viral infection, and this was reversible upon 6 mo of viral suppression with IFN-free HCV therapy. These results implicate that IL-7-dependent NK cell activation and effector function may be other host immune surveillance mechanisms that are impaired in viral infections.
© Society for Leukocyte Biology.

NK cells have shown promise in therapy of hematological cancers, in particular against acute myeloid leukemia. In contrast, the more NK cell-resistant acute lymphoblastic leukemia (ALL) is difficult to treat with NK-cell-based therapies, and we hypothesized that pre-activation of NK cells could overcome this resistance. We show in pediatric and adult patients with T-cell ALL (T-ALL) perturbed NK cell effector functions at diagnosis. Using an in vivo rat model for T-ALL, Roser leukemia (RL), suppressed NK cell effector functions were observed. NK cells from T-ALL patients had reduced expression of the activating receptors NKp46 and DNAM-1, but not NKG2D. In contrast to T-ALL patients, NKG2D but not NKp46 was downregulated on NK cells during rat RL. Decreased frequencies of terminally differentiated NKG2A+CD57-CD56dim NK cells in human T-ALL was paralleled in the rat by reduced frequencies of bone marrow NK cells expressing the maturation marker CD11b, possibly indicating impairment of differentiation during leukemia. RL was highly resistant to autologous NK cells, but this resistance was overcome upon pre-activation of NK cells with IL-12, IL-15, and IL-18, with concomitant upregulation of activation markers and activating receptors. Importantly, adoptive transfers of IL-12, IL-15, and IL-18 pre-activated NK cells significantly slowed progression of RL in vivo. The data thus shows that T-ALL blasts normally resistant to NK cells may be targeted by cytokine pre-activated autologous NK cells, and this approach could have potential implications for immunotherapeutic protocols using NK cells to more efficiently target leukemia.

Flow cytometric immunophenotpying (FCI) of cerebrospinal fluid (CSF) and other paucicellular fluids has been demonstrated to have increased sensitivity in detection of lymphoma and leukemia when compared to cytomorphology [(1) de Graaf et al., Cytometry Part B 2011, 80B:271-281; (2) Szamosi et al., CLSI Document H56-A-Body Fluid Analysis for Cellular Composition; Approved Guideline, Wayne, PA: Clinical and Laboratory Standards Institute, 2006; (3) Kraan et al., Flow Cytometric Immunophenotyping of Cerebrospinal Fluid. Current Protocols in Cytometry, Hoboken, NJ: Wiley, 2008]. However, low cellularity has been an historical problem with these samples. Several studies indicate that immediate addition of a stabilization media (e.g., RPMI with fetal calf serum (FCS)) to CSF improves the cell yield for FCI [(1) de Graaf et al.]. Such stabilization medias can, however, significantly increase cost.
We compared FCI results in CSF stabilized with RPMI 1640 (without additional additives) to results obtained using non-stabilized CSF. Samples were processed according to published CLSI guidelines [(2) Szamosi et al.].
About 98/105 (93%) CSF specimens stabilized with RPMI had adequate numbers of viable cells (>100) for performing FCI. About 65/217 (30%) CSF specimens without stabilization had adequate numbers of viable cells for analysis (70% either quantity not sufficient (QNS) or specimen viability below analytical limits).
Utilizing RMPI without FCS as a stabilization media results in increased cell yield and improved FCI results. We have found FCS is not required to achieve high quality results in FCI of paucicellular CSF specimens.
Copyright © 2013 International Clinical Cytometry Society.