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Originally published online as doi:10.1189/jlb.0906549 on December 8, 2006

Published online before print December 8, 2006
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(Journal of Leukocyte Biology. 2007;81:632-641.)
© 2007 by Society for Leukocyte Biology

Optimization of a myeloid cell transfusion strategy for infected neutropenic hosts

Brad J. Spellberg*,{dagger},1, Mary Collins*,2, Valentina Avanesian*, Mayela Gomez*,{ddagger}, John E. Edwards, Jr.*,{dagger}, Christopher Cogle§, David Applebaum||, Yue Fu*,{dagger} and Ashraf S. Ibrahim*,{dagger}

* Division of Infectious Diseases and
|| Department of Radiation Safety, Los Angeles Biomedical Research Institute, Harbor-University of California at Los Angeles Medical Center, and
{dagger} The David Geffen School of Medicine at UCLA, Torrance, California, USA;
{ddagger} California State University at Dominguez Hills, Carson, California, USA; and
§ University of Florida Shands Cancer Center, Gainesville, Florida, USA

1 Correspondence: Division of Infectious Diseases, Harbor-UCLA Medical Center, 1124 West Carson St., Torrance, CA 90502, USA. E-mail: bspellberg{at}labiomed.org

ABSTRACT

Although granulocyte transfusion is a logical, therapeutic option for neutropenic patients with refractory infections, significant technical barriers have prevented its widespread use. A novel phagocyte transfusion strategy has been developed based on activation of a human myeloid cell line HL-60. To further define the potential for HL-60 cells to recapitulate white cell transfusions, a shortened duration of activation was evaluated, facile quality control markers were defined, and the impact of low-dose irradiation on cell function was determined. Three days of activation resulted in increased cell viability and in vitro candidacidal capacity but with slightly higher cell replication compared with 7 days of activation. Cell viability and several flow cytometric measurements were accurate, quality control markers for HL-60 activation. In combination with activation, low-dose irradiation abrogated replication while sparing the candidacidal effects of the HL-60 cells. Infusion of irradiated, activated HL-60 cells improved survival of neutropenic, candidemic mice significantly. In summary, activated, irradiated HL-60 cells are microbicidal, have virtually no replicative capacity, and are safe and effective at protecting neutropenic mice against an otherwise 100% fatal candidal infection. With continued development, this strategy to recapitulate neutrophil functions has the potential to serve as an effective alternative to granulocyte transfusions.

Key Words: phagocyte • neutrophil • Candida

INTRODUCTION

Disseminated candidiasis is a common, opportunistic infection in neutropenic patients [1 2 3 4 5 6 ], causing an unacceptable, 50% mortality, despite antifungal therapy [7 8 9 10 11 ]. As survival of neutropenic patients with hematogenously disseminated fungal infections is linearly related to the patients’ granulocyte counts [12 , 13 ], exogenous replacement of phagocytes is of great therapeutic potential for these infections. Although neutrophil transfusions have shown promising results [14 , 15 ], they fell out of favor in the early 1980s as a result of several technical difficulties [16 17 18 19 ], which include the facts that harvesting sufficient neutrophils to mediate a protective effect (≥1.5x109 neutrophils/kg/day in infected patients [20 ]) is difficult to achieve [19 ]; ex vivo neutrophils rapidly undergo apoptosis and quickly lose their ability to chemotax to and kill microorganisms [19 ], particularly for killing of Candida, as compared with smaller bacterial organisms [21 ]; and RBCs, lymphocytes, platelets, and/or donor alloantibodies inevitably contaminate even the most pure neutrophil harvests [22 , 23 ], so the donor pool is limited by the need to cross-match blood types, and neutropenic recipients may be at risk for graft-versus-host disease or cytomegalovirus infection from transfused lymphocytes.

To garner the therapeutic benefit of neutrophil transfusions but avoid the technical obstacles mentioned above, we have found that a human, immortal, phagocytic cell line, HL-60, is capable of recapitulating neutrophil host-defense functions in vitro and in vivo in candidemic, neutropenic mice [24 ]. The HL-60 cell line was originally obtained by leukapheresis of peripheral blood cells from a patient with acute myelogenous leukemia [25 ] and has been maintained in culture without genetic manipulation since that time [package insert, American Type Culture Collection (ATCC), Manassas, VA, USA]. A major advantage of HL-60 cells is that they can be activated to differentiate toward a mature neutrophil phenotype [26 ]. Specifically, when activated for 7 days in the presence of retinoic acid (RA) and DMSO, we found a significant improvement in HL-60 cell phagocytosis and killing of the pathogenic fungus, Candida albicans in vitro [24 ]. Furthermore, when administered to neutropenic mice hematogenously infected with C. albicans, activated HL-60 cells markedly improved survival compared with mice treated with control. Immunohistochemistry confirmed that activated HL-60 cells chemotaxed into infected tissues, and histology demonstrated direct phagocytosis of C. albicans in tissues by HL-60 cells [24 ]. Survival was improved, and tissue fungal burden was reduced only by viable HL-60 cells and not nonviable cells. These data indicated that the mechanism of protection was in vivo phagocytosis and clearance of the fungus from infected tissues by viable HL-60 cells.

Our prior studies relied on a 7-day period of HL-60 cell activation, which had several deficits, including the impracticality of waiting 7 days to deliver cells to an infected, neutropenic host and the fact that ≥90% of the cells died during the activation period. As a result of the decline in cell viability over 7 days, enormous quantities of tissue culture materials were required to grow sufficient cells to treat mice. These large amounts of tissue culture materials would make impractical any future clinical application of HL-60 cells, especially considering that ~3500 times more cells would be required per dose on a mg/kg basis (20 g mouse vs. 70 kg adult patient).

To further define the potential using HL-60 cell transfusions to recapitulate neutrophil host defense functions, we now describe significant modifications to the strategy that result in a shortened duration of activation, a marked increase in cell viability during activation, a virtually complete abrogation of HL-60 cell replication, and the use of facile quality control markers to check the adequacy of activation from batch to batch.

MATERIALS AND METHODS

Culture, activation, harvesting, and irradiation of cells
HL-60 cells (ATCC) were cultured at 37°C in 5% CO2 in RPMI 1640, supplemented with glutamine (Irvine Scientific, Santa Ana, CA, USA), 10% FBS (Gemini BioProducts, Woodland, CA, USA), 1% penicillin, streptomycin, and glutamine (Gemini BioProducts), and 50 µM ß-ME (Sigma-Aldrich, St. Louis, MO, USA). HL-60 cells were activated by incubation in the presence of 1.3% (v/v) DMSO and 2.5 µM RA (both from Sigma-Aldrich). For harvesting, cells were centrifuged at 250 g, washed in PBS (Irvine Scientific), and resuspended at the appropriate concentration. The conditioned media of activated HL-60 cell cultures was washed in parallel for use as a placebo for in vivo treatment. To wash the conditioned media, it was centrifuged and decanted, and PBS was added to the residua at an equivalent volume to the final HL-60 cell suspension.

In some experiments, HL-60 cells were irradiated with high-energy X-rays by exposure to 137cesium chloride in a JL Sheppard and Associates (San Fernando CA, USA) irradiator (Model 143-45) in the Clinical Blood Bank Laboratory at Harbor-UCLA Medical Center (Torrance, CA, USA). Radiation doses were confirmed using thermoluminescent dosimetry chips (Global Dosimetry Solutions, Inc., Irvine, CA, USA).

For murine infections, C. albicans SC5314 [24 , 27 ], a well-characterized, clinical isolate, which is highly virulent in animal models, was serially passaged three times in yeast peptone dextrose broth (YPD; Difco, Detroit, MI, USA) and washed twice with PBS. Infectious inocula were prepared by counting in a hemacytometer and confirmed by colony counting.

In vitro killing and proliferation assays
To quantify the candidacidal effect of HL-60 cells, our killing assay described previously was used [24 ]. In brief, 8 x 104 phagocytes were cocultured with 4 x 103 C. albicans (20:1 ratio) for 4 h at 37°C in RPMI + 10% pooled human serum (Sigma-Aldrich). At the end of the incubation, the cultures were sonicated, serially diluted, overlaid with YPD agar, and incubated overnight at 37°C. CFUs were counted to assess killing of C. albicans compared with control cultures of Candida alone with no HL-60 cells.

To quantify cell proliferation, tritiated thymidine (Amersham Biosciences, Irvine, CA, USA) was added to HL-60 cell cultures. Eighteen hours later, the cells were harvested on filter paper using a Combi cell harvester (Molecular Devices, Sunnyvale, CA, USA). The filter paper was dissolved in scintillation fluid, and radioactive counts were determined in a ß-counter.

Oxidative burst measurement and flow cytometry
To determine the degree of activation of HL-60 cells, a modification of the method of Roesler et al. [28 , 29 ] was used. This technique relies on the induction of fluorescence of dihydrorhodamine (DHR) in the presence of reactive oxygen intermediates and is used clinically to diagnose chronic granulomatous disease. In brief, unactivated or activated HL-60 cells were incubated in the dark at 37°C for 20 min in the presence or absence of 50 µg/ml PMA (Sigma-Aldrich) and 100 µM DHR 123 (Molecular Probes, Eugene, OR, USA). The cells were then analyzed for size and fluorescence with a FACSCalibur II instrument (Becton Dickinson, San Jose, CA, USA) using FACSComp software per the manufacturer’s recommendations. During acquisition, photomultiplier amplitudes were set based on negative control cells, which were cultured without PMA (unstimulated), and with or without DHR. Preliminary investigations determined that DHR fluorescence in channels FL1 or FL2 were similar for HL-60 cells; hence, FL1 was used for subsequent evaluations.

Mice and survival experiments
Male BALB/c mice (20–25 g) were obtained from the National Cancer Institute (Frederick, MD). Mice were made neutropenic by a single i.p. injection of cyclophosphamide (230 mg/kg) [30 ]. In pilot studies in which peripheral blood leukocytes were manually counted, this treatment regimen resulted in pancytopenia from Days 1 through 7 postcyclophosphamide treatment (Days 0–6 postinfection given infection on Day 1 postcyclophosphamide), and recovery of cell counts began on Day 8 postcyclophosphamide (Day 7 postinfection). Mice were infected via the tail vein with 0.2 ml PBS containing 5 x 104 blastoconidia of C. albicans ~32 h after cyclophosphamide injection, as described previously [27 ].

Treatment with 1.5 x 107 HL-60 cells/mouse (~7.5x108 cells/kg) or placebo in 0.25 ml PBS was administered via the tail vein 30–60 min after infection on Day 0 and again on Days 2, 4, and 6 postinfection. All procedures involving mice were approved by the Institutional Animal Use and Care Committee, following the National Institutes of Health guidelines for animal housing and care.

Statistics
Kaplan-Meier curves were compared pair-wise with the nonparametric log rank test. In vitro killing and replication and in vivo tissue fungal burden were compared with the nonparametric Steel test for multiple comparisons [31 ] or the Wilcoxon rank sum test as appropriate.

RESULTS

HL-60 cells are not infected with common viral pathogens
As a marker of safety, HL-60 cells and conditioned HL-60 cell supernatant were tested commercially (Focus Laboratories, Cypress, CA, USA) for a variety of human pathogenic viruses. PCR testing was negative for human T lymphotrophic virus (HTLV)-1 and HTVL-2, hepatitis C virus (HCV; by RT-PCR), HBV, HIV (by ultrasensitive RT-PCR), human herpes virus (HHV-6) and HHV-7, varicella zoster virus, and EBV.

A shortened duration of activation of HL-60 cells resulted in the equivalent of superior killing of C. albicans
We have reported previously that activation of HL-60 cells for 7 days in the presence of RA and DMSO resulted in superior fungicidal activity compared with activation with either compound alone [24 ]. To determine if shorter periods of dual activation similarly stimulated HL-60 fungicidal effects and improved viability, we activated HL-60 cells for 1, 3, or 7 days in RA plus DMSO. All three activation strategies resulted in superior killing of C. albicans by HL-60 cells as compared with unactivated HL-60 cells (Fig. 1 A). Activation for 3 days resulted in superior killing compared with all other groups. Given the ≥90% loss in HL-60 cell viability following 7-day activation [24 ] and the impracticality of waiting 7 days for activation to occur, further investigations focused on the 1- and 3-day activation strategies.


Figure 1
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  		Figure 1. Three days of HL-60 activation optimizes killing of C. albicans and results in sufficient cell differentiation to distinguish unactivated cells. HL-60 cells were activated with RA and DMSO for 1, 3, or 7 days (1 D, 3 D, 7 D, respectively). (A) Unactivated or activated HL-60 (8x104) was incubated with C. albicans (4x103) at a 20:1 ratio for 4 h. Results are from a minimum of three separate experiments run in triplicate. *, P < 0.05, versus unactivated; {ddagger}, P < 0.007, versus all other groups by Steel test for nonparametric multiple comparisons. (B) Viability determined by trypan blue exclusion. n = 22 samples per group. Top and bottom bars reflect 95% confidence interval; middle bar is the mean. *, P < 0.0001, compared with unactivated (U) and 1-day-activated by Student’s t test. (C) Size of cells determined by forward/side-scatter (FSC). (D) Oxidative burst as determined by DHR fluorescence in the absence (Unstimulated) or presence (Stimulated) of PMA. Results are representative of six experiments.

 
Viability and several flow cytometric parameters reliably distinguished unactivated from activated HL-60 cells
Given the need to ensure adequate activation of HL-60 cells from experiment to experiment, several cellular parameters were explored as potential quality control markers of activation. Concordant with our prior results using 7-day-activated cells [24 ], we found that continuous activation of the cells in the presence of RA and DMSO resulted in differentiation of the cells and a progressive decrease in cell viability starting after Day 1 of activation. By Day 3 of activation, HL-60 cell viability was decreased significantly and reliably as compared with unactivated and 1-day-activated cells (Fig. 1B) .

During cell counting, we noted that activated HL-60 cells appeared smaller than unactivated HL-60 cells. This significant change in size was confirmed by flow cytometry (FSC) for 3-day- but not 1-day-activated HL-60 cells compared with unactivated cells (Fig. 1C ; Table 1 ).


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  		Table 1. Quality Control Results

 
We also quantified the oxidative burst capacity of unactivated and activated HL-60 cells in the presence or absence of PMA by using DHR fluorescence. Prior to PMA stimulation, unactivated HL-60 cells had significantly greater oxidative activity (reflected by greater DHR fluorescence) than did 3-day-activated but not 1-day-activated HL-60 cells (Fig. 1D ; Table 1 ). In contrast, PMA stimulation resulted in a marked increase in oxidative activity (i.e., increased DHR fluorescence) in 3-day-activated HL-60 cells but not in unactivated or 1-day-activated HL-60 cells (Fig. 1D ; Table 1 ).

Several flow cytometric parameters correlated with HL-60 cell candidacidal capacity
To determine if parameters that distinguished unactivated from activated HL-60 cells correlated with candidacidal capacity, cellular parameters were measured immediately before performing kill assays with the same cells. Consistent with HL-60 cell maturation during activation, decreasing cell viability, decreasing cell size, and change in cell size during activation each correlated with anticandidal activity, regardless of whether all groups were compared, or only unactivated versus 3-day-activated cells were compared (Fig. 2 ).


Figure 2
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  		Figure 2. HL-60 viability and cell size correlate with anticandidal killing activity. Immediately prior to performing anti-C. albicans kill assays, viability (trypan blue exclusion) and flow cytometry parameters were determined on unactivated HL-60 cells or HL-60 cells activated with RA and DMSO for 1 or 3 days. Size ratio indicates the size of 1- or 3-day-activated cells divided by the size of unactivated cells. Results are from six experiments, and each data point reflects the mean of triplicate values from each experiment. {rho} and P values were determined by the nonparametric Spearman correlation test.

 
Of note, the mean DHR fluorescence intensity (reflecting oxidative activity) of HL-60 cells prior to PMA stimulation correlated inversely with killing ({rho}=–0.8 and P=0.002 for all groups; {rho}=–0.7 and P=0.02 for unactivated vs. 3-day-activated), as did the percent of fluorescent cells ({rho}=–0.5 and P=0.04 for all groups; {rho}=–0.5 and P=0.08 for unactivated vs. 3-day-activated; Fig. 3 ). In contrast, following PMA stimulation, the change in fluorescence ({rho}=0.8 and P=0.002 for all groups; {rho}=0.8 and P=0.01 for unactivated vs. 3-day-activated) and change in percent fluorescent cells ({rho}=0.8 and P=0.002 for all groups; {rho}=0.6 and P=0.04 for unactivated vs. 3-day-activated) correlated positively with anticandidal killing (Fig. 3) .


Figure 3
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  		Figure 3. Modified phagoburst activity correlates with anticandidal killing of HL-60 cells. Median fluorescence intensity (MFI) in channel FL1 and percent fluorescent cells were determined by flow cytometry of unstimulated cells (i.e., no PMA exposure, reflecting baseline oxidative burst activity). Stimulation index (SI) refers to the ratio of fluorescence of HL-60 cells after stimulation with PMA divided by the fluorescence of unstimulated cells. Results are from six experiments, and each data point reflects the mean of triplicate values from each experiment. {rho} and Pvalues were determined by the nonparametric Spearman correlation test.

 
Irradiation abrogated HL-60 cell replication without impairing the anticandidacidal capacity of the cells
To mitigate any long-term risk of infusing an immortal cell line into a neutropenic host, we determined the impact of irradiation on cell replication and anticandidal capacity. Radiation doses were chosen initially based on those used clinically for blood products being infused into myeloablated patients. Irradiation with 2100 ± 300 rads resulted in a marked decrease in cell replication in unactivated and in 1- or 3-day-activated HL-60 cells (Fig. 4 A). This decline in replication became progressively greater between Days 1 and 3 following the irradiation. In contrast, 7-day-activated HL-60 cells already had a low, replicative capacity, which was not lowered significantly further by irradiation.


Figure 4
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  		Figure 4. Irradiation abrogates replication and sparing anticandidal effects of HL-60 cells, which were activated with RA and DMSO for 1, 3, or 7 days. Unactivated (Unact.) or activated cells were then irradiated with 2100 ± 300 rads. Kill assays were performed with irradiated or unirradiated cells. The same batches of cells were cultured for an additional 24, 48, or 72 h following irradiation prior to determination of replication activity. Results are from four experiments, performed in duplicate for replication or triplicate for killing. {ddagger}, P < 0.05, versus unactivated, unirradiated cells and background; *, P < 0.05, versus unactivated, unirradiated cells by the nonparametric Steel test for multiple comparisons.

 
Irradiation had no significant impact on the anticandidacidal effects of the HL-60 cells (Fig. 4B) . Irradiation also had no impact on the accuracy of cell size or cell fluorescence as quality control markers for successful activation of HL-60 cells (Fig. 5 ).


Figure 5
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  		Figure 5. Irradiation (I) does not alter the accuracy of several quality control markers of HL-60 cell activation. MFI in channel FL1 and percent fluorescent cells were measured in HL-60 cells. Ratio refers to size of HL-60 cells activated with RA and DMSO, divided by the size of unactivated HL-60 cells. Results are from three experiments, and percent killing reflects the mean of triplicate values from each experiment. {rho} and P values were determined by the nonparametric Spearman correlation test. UU, Unactivated, unirradiated.

 
Activated, irradiated HL-60 cells improved survival of neutropenic, candidemic mice by reducing tissue fungal burden
To determine if the shortened activation/irradiation regimen resulted in HL-60 cells, which could treat candidemia in a neutropenic host, mice were made neutropenic with cyclophosphamide and infected via the tail vein with C. albicans. Mice were treated with placebo, or 1- or 3-day-activated HL-60 cells, which were irradiated or unirradiated. Treatments were administered i.v. via the tail vein, starting 1 h after infection and again on Days 2, 4, and 6 after infection. Mice treated with irradiated, 1- or 3-day-activated HL-60 cells had improved survival significantly compared with all other treatment regimens (Fig. 6 A). However, only treatment with 3-day-activated, irradiated HL-60 cells resulted in long-term survival.


Figure 6
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  		Figure 6. Activated, irradiated HL-60 cells improved the survival of and reduced tissue fungal burden in candidemic, neutropenic mice. (A) Male BALB/c mice (n=10 from two experiments) were made neutropenic with cyclophosphamide and infected via the tail vein with 5 x 104 blastospores of C. albicans. HL-60 cells were administered i.v. via the tail vein on Days 0, 2, 4, and 6 postinfection. The combined experiment is broken into two graphs for ease of display; the placebo curve is shown in both graphs for ease of comparison. *, P < 0.05, versus all groups except 3-day-activated, unirradiated and 3-day-activated, irradiated; **, P < 0.05, versus all groups except 1-day-activated, irradiated (P=0.16) by log rank test. (B) Day 5 kidney fungal burden (n=8 mice per group) in candidemic, neutropenic mice treated with HL-60 cells or placebo on Days 0, 2, and 4 postinfection. y-Axis reflects lower limit of detection of the assay. *, P < 0.05, versus placebo by nonparametric Steel test for multiple comparisons.

 
To confirm that activated, irradiated HL-60 cells enhanced clearance of the fungus from neutropenic mice, we determined tissue fungal burden in mice treated with placebo or 1- or 3-day-activated, irradiated HL-60 cells. Neutropenic mice were infected and treated as above; however, on Day 5 postinfection (after three treatment doses, on the day when placebo-treated mice were anticipated to begin dying), mice were killed, and their kidneys were homogenized and cultured quantitatively. Kidneys were used, as they are the predominant target organ of infection during murine candidemia [24 , 27 , 32 33 34 35 36 ]. Treatment with 1- or 3-day-activated, irradiated HL-60 cells resulted in ~0.7 log CFU/g reduction in kidney fungal burden (Fig. 6B) .

To determine if HL-60 cell therapy resulted in any end-organ toxicities, tissue histopathology was evaluated on Day 5 postinfection, after three doses of HL-60 cells were administered on Days 0, 2, and 4 postinfection. A variety of organs was evaluated, including brain, heart, lung, sternum (for bone marrow), spleen, liver, and kidneys. Compared with mice treated with placebo, there was no discernable toxicity found in any organ (data not shown).

DISCUSSION

We have developed a novel, cell-based immunotherapy, which recapitulates neutrophil functions in neutropenic, candidemic mice. Administration of activated, irradiated HL-60 cells resulted in enhanced clearance of tissue fungal burden and improved survival. In a previous study, we confirmed that activated HL-60 cells chemotaxed into infected tissues and phagocytized C. albicans, lowering fungal burden [24 ]. However, 7 days of activation resulted in ≥90% decreases in HL-60 cell viability, requiring impractical quantities of culture media. The increased viability of cells after a 3-day activation protocol makes the strategy practical for future clinical use. For example, scale-up in a mid-sized industrial fermenter (i.e., 10,000 L reactor) would allow production every 72 h of sufficient cells for at least 200 doses of 1.5 x 109 cells/kg for 70 kg adults (2x106 cells/mlx103 ml/literx104 liters=2x1013 cells=200 doses of 1x1011 cells).

Our current culture technique in 400 ml tissue-culture flasks (which hold 250 ml vol) affords a significantly smaller capacity. It must be emphasized that as a result of the limitations of culture media required, our studies to date have used treatment doses of phagocytes that are ≥50% below the ~1.5 x 109 cells/kg target dose, which would mimic the normal, endogenous turnover rate in infected hosts [20 , 37 38 39 40 ]. The current studies with 3-day-activated cells used this same dose to allow an approximate comparison with our previously reported efficacy with 7-day-activated cells. However, the increased viability of 3-day-activated cells will allow dose escalation studies to optimize the protective effect in future studies.

The results of the survival studies paralleled the replication data, indicating that continued replication in HL-60 cells mitigates any protective effect. Specifically, 7-day activation of HL-60 cells resulted in a virtual, complete abrogation of HL-60 cell replication, as did irradiation of 3-day-activated HL-60 cells. Activation for shorter periods of time reduced replication significantly but did not abrogate it completely. Irradiated, 1-day-activated cells had replication rates that were significantly higher than irradiated, 3-day-activated cells. Paralleling these replication data, survival studies demonstrated that only treatment of candidemic, neutropenic mice with irradiated, 3-day-activated HL-60 cells resulted in long-term survival, as did 7 days of activation in our prior study [24 ]. Administration of 1- or 3-day-activated but unirriadiated HL-60 cells did not improve survival, despite the fact that the cells were highly active in in vitro killing assays. Collectively, these data indicate that two factors are required for HL-60 cell transfusions to mediate improved survival: enhanced efficacy engendered by activation and abrogation of replication by 7-day activation or by shorter activation periods with irradiation. Abrogation of replication is presumably required to mitigate toxicity of the cells. Indeed, our prior studies demonstrated convincingly that unactivated HL-60 cells were toxic to mice, whereas cells activated for 7 days were not [24 ]. Despite intensive investigation to identify an organ-specific toxicity mediated by replicating HL-60 cells, no specific toxicity could be found by organ histopathology. Investigation into the precise nature of the toxicity of HL-60 cells is ongoing.

We identified several facile markers that reliably distinguished activated from unactivated HL-60 cells. These markers, including cell viability, cell size, and oxidative-free radical formation, correlated with in vitro killing of C. albicans, making them useful quality control markers of activation. The correlations of candidal killing with lower HL-60 cell viability, smaller cell size, and increased oxidative-free radical formation following PMA activation are consistent with maturation of immature HL-60 cells toward a more mature, granulocytic phenotype during activation.

It has been reported previously that differentiation of HL-60 cells with DMSO results in a steady acquisition of PMA-induced, oxidative burst capacity [41 , 42 ]. However, surprisingly, we found that in the absence of PMA, undifferentiated HL-60 cells had greater capacity to produce oxygen-free radicals than differentiated HL-60 cells. This production of oxygen-free radicals by undifferentiated HL-60 cells was likely the result of dysregulated metabolism, as the free-radical production was actually suppressed by stimulation of the cells with PMA. In contrast, normal phagocytes do not produce oxygen-free radicals in the resting state and only do so after stimulation with a cell activator, such as PMA or fMLP. Akin to normal phagocytic function, differentiation of HL-60 cells with DMSO plus RA suppressed oxygen-free radical production completely at baseline but enabled a massive increase in oxygen-free radical production when the cells were stimulated with PMA.

In summary, a combination of low-dose irradiation with dual RA + DMSO activation of HL-60 cells resulted in a marked increase in candidacidal capacity, a virtually total abrogation of cell replication, and a marked improvement in the survival of neutropenic, candidemic mice. Continued investigation to optimize the treatment dose, discern the in vivo half-life of the cells, better understand the toxicity of unactivated/unirradiated HL-60 cells, and determine the feasibility of cryopreservation of preactivated HL-60 cells is ongoing. With continued development, activated, irradiated HL-60 cells have promised to be developed into a practical, rapid, cell-based immunotherapy for refractory infections in neutropenic patients.

ACKNOWLEDGEMENTS

This work was supported by National Institutes of Allergy and Infectious Diseases/National Institutes of Health grants R03 AI054531 (A. S. I.), R01 AI19990 (J. E. E.), and K08 AI060641 (B. J. S.), as well as American Heart Beginning Grant-in-Aid 0665154Y (B. J. S.) and a seed grant from the Los Angeles Biomedical Research Institute (B. J. S.). J. E. E. is also supported by an unrestricted grant for infectious diseases from Bristol Myers Squibb. B. J. S., J. E. E., Y. F., and A. S. I. hold a patent related to the work described. This work was presented in part at the 14th Congress of the Immunocompromised Host Society in Crans-Montana, Switzerland, July 2006.

FOOTNOTES

2 Current address: New York University, 550 First Ave., Smilow Building, Rm. 307, New York, NY 10011, USA. Back

Received September 5, 2006; revised October 27, 2006; accepted November 10, 2006.

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A. S. Ibrahim, T. Gebremariam, Y. Fu, J. E. Edwards Jr., and B. Spellberg
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