T-cell apoptosis induced by granulocyte colony-stimulating factor is associated with retinoblastoma...

Experimental Hematology 29 (2001) 401–415

0301-472X/01 $–see front matter. Copyright © 2001 International Society for Experimental Hematology. Published by Elsevier Science Inc.PII S0301-472X(01)00617-8

T-cell apoptosis induced by granulocyte colony-stimulating factor is associated with retinoblastoma protein

phosphorylation and reduced expression of cyclin-dependent kinase inhibitors

Sergio Rutella

a

, Luca Pierelli

a

, Carlo Rumi

a

, Giuseppina Bonanno

b

, Maria Marone

b

,Simona Sica

a

, Ettore Capoluongo

c

, Franco Ameglio

c

, Giovanni Scambia

b

, and Giuseppe Leone

a

a

Department of Hematology and

b

Department of Gynecology, Catholic University Medical School, Rome, Italy; and

c

Department of Clinical Pathology, IRCCS San Gallicano, Rome, Italy

(Received 28 September 2000; revised 16 November 2000; accepted 29 November 2000)

Objective.

Peripheral blood progenitor cells (PBPC) mobilized by granulocyte colony-stimu-lating factor (G-CSF) promptly engraft allogeneic recipients after myeloablative chemother-apy for hematologic malignancies. Surprisingly, no exacerbation of acute graft-vs-host diseasehas been observed despite a 10-fold higher T-cell content in PBPC compared with bone mar-row allografts. Because G-CSF can suppress T-cell proliferation in response to mitogens andenhance their activation-induced apoptosis, we examined the molecular mechanisms underly-ing G-CSF–induced immune dysfunction.

Materials and Methods.

Normal allogeneic lymphocytes were challenged with phytohemagglu-tinin in the presence of serum collected after G-CSF administration (postG) to healthy PBPCdonors, and the expression of key components of the cell cycle and apoptotic machineries wasinvestigated by flow cytometry and Western blotting.

Results.

Lymphocyte stimulation was associated with collapse of mitochondrial transmem-brane potential, hypergeneration of reactive oxygen intermediates, and activation of caspase-3

and DNA fragmentation. Lymphocytes were arrested in a G

1

-like phase of the cell cycle, asmeasured by G

1

-phase cyclin expression and bromodeoxyuridine (BrdUrd) incorporation.Cell tracking experiments confirmed the occurrence of a lower number of population dou-blings in postG compared with preG cultures. Unexpectedly, the phosphorylation state of theprotein encoded by the retinoblastoma susceptibility gene (pRB) was unaltered in postG cul-tures, and the inhibition of cell cycle progression occurred without the recruitment of the cy-clin-dependent kinase inhibitors p15

INK4B

, p16

INK4A

, and p27

Kip1

. We eventually evaluated theability of antioxidant/cytoprotectant agents to prevent the G-CSF–induced mitochondrial dys-function and inhibition of cell cycle progression. Of interest, both

N

-acetylcysteine and amifos-tine reduced apoptotic cell death by 45% on average, inhibited the activation/processing ofcaspase-3, and increased BrdUrd incorporation in postG cultures.

Conclusions.

Based on these experimental findings, a model is proposed in which T-cell acti-vation in the presence of serum immunoregulatory factor(s) induced by G-CSF is associatedwith a molecular phenotype mimicking the G

1

–S transition and consisting of pRB phosphory-lation, lack of CDKI recruitment, and reduced cyclin-E expression. The putative relationshipbetween lymphocyte mitogenic unresponsiveness and apoptosis induction would occur at thelevel of key molecules shared by the cell cycle and apoptotic machineries. Whether theG-CSF–mediated modulation of lymphocyte functions in vitro is beneficial in transplantationmedicine remains to be determined. © 2001 International Society for Experimental Hematology.

Published by Elsevier Science Inc.

Offprint requests to: Sergio Rutella, M.D., Department of Hematology, Catholic University Medical School, Largo A. Gemelli 8-00168 Rome, Italy; E-mail:[email protected]

402

S. Rutella et al./Experimental Hematology 29 (2001) 401–415

Introduction

Recombinant human granulocyte colony-stimulating factor(G-CSF) can be administered to human leukocyte antigen(HLA)-identical as well as to HLA-A, HLA-B, and HLA-DRcompatible unrelated donors to mobilize peripheral blood pro-genitor cells (PBPC) for subsequent transplantation into allo-geneic recipients [1,2]. Contrary to expectations, incidenceand severity of acute graft-vs-host disease (aGVHD) were notincreased after PBPC compared with bone marrow allogeneictransplantation, despite a 10-fold higher T-cell content inG-CSF–mobilized grafts [3–5]; however, the incidence ofclinical extensive chronic GVHD might be increased [6].

The regulatory effects of G-CSF on T lymphocytes werereviewed recently [7,8]. Several investigations suggest a hu-moral modulation of T-cell function both in animal modelsand in humans, which consists of 1) downregulation of Thelper-1 (Th1)-derived cytokines and polarization toward aTh2 functional profile [9]; 2) increase of serum immunoreg-ulatory mediators, i.e., lactoferrin, interleukin-1 receptor an-tagonist (IL-1ra), and IL-10 [10,11]; 3) lymphocyte unre-sponsiveness; and 4) induction of lymphocyte partialactivation after mitogenic challenge [12,13]. However, ad-ditional mechanisms of immune suppression by G-CSFhave been claimed, i.e., monocyte-mediated inhibition ofT-cell proliferation through CD28 responsive complex, aninducible T-cell transcription factor binding in the promoterof the interleukin-2 (IL-2) gene [14–16].

We recently described an enhanced tendency to activa-tion-induced apoptosis of allogeneic CD4

1

and CD8

1

Tcells stimulated with phytohemagglutinin (PHA) in the pres-ence of serum collected after G-CSF administration [17].Conceivably, those phenomena were mediated by Bax pro-tein overexpression with subsequent collapse of mitochon-drial transmembrane potential, hypergeneration of reactiveoxygen intermediates (ROIs), and fragmentation of genomicDNA. The neutralization of surface CD95 abrogated the per-turbation of mitochondrial function, strongly suggesting theinvolvement of the CD95–CD95L signaling pathway [17].

The mitogen-dependent progression through the G

1

phaseof the cell cycle and the initiation of DNA synthesis are co-operatively regulated by the cyclin-dependent kinases(CDKs), whose activity is controlled by their associationwith cyclins and CDK inhibitors (CDKI) [18]. Upon stimu-lation with mitogens, the protein encoded by the retinoblas-toma susceptibility gene (pRB) is inactivated by phosphory-lation mediated by the sequential intervention of D-typecyclins and cyclin E. Hyperphosphorylation renders pRBincapable of binding E2F-type transcription factors, leadingto the activation of genes necessary for DNA synthesis andfor the advancement through the cell cycle [19–21].

Experimental evidence supports an interconnection be-tween cell cycle and apoptosis, a ubiquitous physiologic pro-cess through which multicellular organisms eliminate dam-aged or unwanted cells. In particular, pRB, D-type cyclins,and CDKI have been proposed as candidate molecules af-

fecting both cell cycle progression and apoptotic cell death[22]. Recently, a novel and intriguing function of mitochon-dria has emerged in apoptosis research, in addition to theirestablished role as energy-producing organelles. In particu-lar, mitochondria might be central executioners of apoptoticprocesses, and both collapse of mitochondrial transmem-brane potential and hypergeneration of ROIs might representgeneral features of programmed cell death [23].

Because the molecular mechanisms underlying lympho-cyte unresponsiveness to mitogenic challenge after exposureto G-CSF–induced soluble immunoregulatory mediators re-main largely unknown [24], we investigated the expressionof key components of the cell cycle and apoptotic machiner-ies in normal allogeneic lymphocytes activated with PHA inthe presence of serum collected after G-CSF administration(postG) to healthy PBPC donors. To this purpose, lympho-cyte mitochondrial function, phosphorylation state of cell cy-cle related proteins (pRB, p107), and expression of G

1

-phasecyclins and representative CDKI (p15

INK4B

, p16

INK4A

,p21

Cip1

, p27

Kip1

) were measured. This study clearly demon-strates that lymphocyte stimulation in the presence of postGserum is associated with molecular events mimicking thetransition through the G

1

–S phase of the cell cycle but ulti-mately leading to the execution of an apoptotic program.

Materials and methods

Characteristics of PBPC donors and collection of donor serum

Six Caucasian HLA-identical healthy donors (three men and threewomen; median age 36 years) received G-CSF (16

m

g/kg/day;Granocyte Rhone-Poulenc Rorer, Milan, Italy) subcutaneously for6 days to mobilize PBPC for allogeneic transplantation. Donor se-rum was collected before (preG) and on day 4 of G-CSF adminis-tration (postG), aliquoted, and stored at

2

80

8

C until use, as de-tailed previously [10,25]. Informed consent was obtained fromPBPC donors, and the investigations were approved by the Institu-tional Human Research Committee.

Preparation of peripheral blood lymphocytes and culture conditions

Heparin-anticoagulated peripheral blood samples from normalG-CSF–untreated subjects were diluted 1:1 in RPMI 1640 culturemedium, layered on a Ficoll-Hypaque gradient (density 1,077 g/L;Uppsala, Sweden), and centrifuged at 1,700 rpm for 30 minutes at20

8

C. Peripheral blood mononuclear cells (PBMC) were harvestedat the interface, washed in RPMI 1640 at 1,500 rpm for 6 minutes,and seeded at a concentration of 1

3

10

6

/mL in RPMI 1640 me-dium either under serum-free (SF) conditions (10% BIT HCC-9500serum substitute; StemCell Technologies, Vancouver, BC, Canada)or with preG or postG serum (20% final concentration, v/v). Cellswere stimulated with PHA (0.5

m

g/mL) for 72 hours at 37

8

C in hu-midified atmosphere (5% CO

2

and 95% air), harvested, counted,and used as described in detail later.

Evaluation of lymphocyte mitochondrial function

The simultaneous assessment of surface markers, mitochondrialinner transmembrane potential (

Dc

m

), and generation of ROIs was


403

performed as follows [17,23]. Cells were stained with phycoeryth-rin (PE)-conjugated anti-CD4 (S3.5 clone, IgG

2a

) or anti-CD8(3B5 clone, IgG

2a

) monoclonal antibodies (mAb) or with PE-con-jugated isotype-matched irrelevant mAb (Caltag Laboratories,Burlingame, CA, USA) for 30 minutes at 4

8

C and then exposed for15 minutes to 3,3

9

-dihexyloxacarbocyanine iodide [DiOC

6

(3), 40nM] and to hydroethidine (HE; 2 mM; Molecular Probes, Eugene,OR, USA) prior to flow cytometric analysis. In control experi-ments, cells were incubated with carbonyl cyanide m-chlorophe-nyl-hydrazone (mClCCP, 50

m

M; Sigma Chemical Corp., St.Louis, MO, USA), an uncoupler of oxidative phosphorylation, at37

8

C for 30 minutes before DiOC

6

(3) labeling. MClCCP treatmentcompletely abolishes

Dc

m

, demonstrating that DiOC

6

(3) uptake isdriven by

Dc

m

and does not involve significant binding to othercellular components. In selected experiments aimed at protectinglymphocytes from activation-induced apoptosis, PBMCs were pre-incubated with

N

-acetylcysteine (NAC; 10 mM/L) or amifostine(60

m

g/mL) for 30 minutes prior to PHA challenge in the presenceof preG or postG serum [26,27].

Detection of processed caspase-3

After PHA activation, cells were treated with fixation medium (so-lution A; Fix & Perm, Caltag Laboratories) and incubated for 15minutes at room temperature. After washings in phosphate-buff-ered saline (PBS) supplemented with 1% bovine serum albumin(BSA), cells were resuspended in permeabilizing medium (solu-tion B) containing saturating amounts of rabbit polyclonal anti-activecaspase-3 antibody (PharMingen, San Diego, CA, USA), followedby incubation with fluorescein isothyocyanate (FITC)-conjugatedgoat anti-rabbit immunoglobulins (Caltag Laboratories) for 30minutes at 4

8

C. The anti-active caspase-3 antibody reacts with aconformational epitope exposed on the processed forms ofcaspase-3 and recognizes cells undergoing apoptosis [28].

Bromodeoxyuridine incorporation assay

Lymphocyte proliferation was analyzed after 24, 48, and 72 hoursof PHA stimulation by evaluating the incorporation of bromodeox-yuridine (BrdUrd), a thymidine analogue, in newly synthesizedDNA strands. Briefly, cells were exposed to BrdUrd (25

m

M finalconcentration; Sigma Chemical Corp.) during mitogenic chal-lenge. Aliquots of cells were harvested at indicated time points andwere fixed with ice-cold 70% ethanol. Because anti-BrdUrd mAbsreact with single-stranded DNA, partial denaturation was achievedby incubation with 3N HCl containing 0.5% Tween-20 for 20 min-utes at room temperature. After washings with PBS, cells were re-suspended in 0.1 M Na

2

B

4

O

7

to neutralize residual HCl and subse-quently incubated with pretitrated dilutions of FITC-conjugatedanti-BrdUrd mAb (BR-3 clone, IgG

1

; Caltag Laboratories) for 30minutes at 4

8

C. Background fluorescence was established with flu-orochrome-conjugated isotype-matched irrelevant immunoglobu-lins. Cells not exposed to BrdUrd during PHA activation wereused to evaluate the specificity of the anti-BrdUrd staining. Cellu-lar DNA content was measured by resuspending the BrdUrd-labeled cells in DNA staining buffer (5

m

g/mL propidium iodide,2 mg/mL RNAse) for 30 minutes prior to flow cytometric analysis.

Detection of G

1

-phase cyclins

PHA-challenged PBMCs were sequentially fixed with 0.1%paraformaldehyde and 70% ethanol, as previously reported [29]. Af-ter washings with PBS containing 0.05% Tween-20, cells were incu-bated overnight with FITC-conjugated anti-D-type cyclin mAb

(G107-565 clone, IgG

1

) or unconjugated anti-cyclin E mAb (HE12clone, IgG

1

; both from PharMingen). Samples labeled with the pri-mary unconjugated anti-cyclin E mAb were further incubated withFITC-conjugated F(ab

9

)

2

goat anti-mouse immunoglobulins (CaltagLaboratories) for 30 minutes at 4

8

C [30]. Controls were preparedidentically as described, except that an isotype-matched mAb wasadded instead of the anti–D-type cyclin and anti-cyclin E mAb. Forthe evaluation of cyclin vs DNA content, cells were resuspended inDNA staining buffer (5

m

g/mL propidium iodide, 2 mg/mL RNAse)and kept on ice for 30 minutes prior to flow cytometric analysis [31].

Detection of cell cycle-related proteins and CDKI

PHA-activated PBMCs were sequentially fixed and permeabilizedas described earlier. After washings in PBS supplemented with 1%BSA, cells were incubated for 30 minutes at 4

8

C with saturatingamounts of antibodies directed against pRB (G3-245 clone, mouseIgG

1

), p107 (SD9 clone, mouse IgG

1

), p16

INK4A

(G175-405 clone,mouse IgG

1

), p21

Cip1

(SX118 clone, mouse IgG

1

), p27

Kip1

(G173-524clone, mouse IgG

1

; all from PharMingen), and p15

INK4B

(sc-611clone, rabbit IgG; Santa Cruz Biotechnology, Santa Cruz, CA,USA). Cells were then washed with PBS supplemented with 1%BSA and labeled with FITC-conjugated F(ab

9

)

2

goat anti-mouse orwith FITC-conjugated goat anti-rabbit immunoglobulins (CaltagLaboratories) for 30 minutes at 4

8

C. Isotypic controls were pre-pared with the appropriate mouse or rabbit irrelevant antibodies.

Western blot analysis

Cell lysate and Western blotting were performed as follows. Thepellet obtained from 1

3

10

6

cells was washed in PBS and dis-solved in lysis buffer (20 nM TRIS HCl at pH 7.4, 0.1 M NaCl, 5 mMMgCl

2

, 1% Nonidet P-40, 0.5% sodium deoxycholate, 2 U/mLkallikrein inhibitor aprotinin, 50 mM NaF, 2 mM Na

3

VO

4

, 1 mMPMSF, 2

m

g/mL leupeptin). Protein concentration was determinedwith the Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules,CA, USA). Thirty micrograms of each protein sample was sepa-rated on 6% polyacrylamide SDS gel and electroblotted on polyvi-nylidene fluoride membranes (Millipore Co., Bedford, MA, USA).Membranes were incubated with 6% nonfat dry milk in 1

3

TBSTfor blocking (0.1 M Trizma base, 0.15 M NaCl, 0.05% Tween-20,pH 7.4) and then with the primary rabbit antibodies in 3% nonfatdry milk in 1

3

TBST (anti-pRB and anti-p107; both from SantaCruz Biotechnology). Following incubation with a goat anti-rabbitsecondary antibody conjugated with horseradish peroxidase (Bio-Rad), detection was performed with the ECL Plus system (Amer-sham International, Buckingamshire, United Kingdom), and theblots were exposed to X-AR-5 OMAT Kodak films. The blotswere reused by stripping at 50

8

C for 30 minutes in 100 mM 2-mer-captoethanol, 2% SDS, and 62.5 mM TRIS HCL, pH 6.7, followedby extensive blocking and reprobing with a different antibody. TheBenchMark prestained protein ladder (Life Technologies Inc.,Rockville, MD, USA) was used as a reference for relative mobil-ity. Images of x-ray films were acquired with a Cohu CCD camera,and quantification of the bands was performed with Photoretix 1D(Photoretix International Ltd., Newcastle Upon Tyne, UnitedKingdom). Band intensity was expressed as relative absorbanceunits.

Measurement of lymphocyte divisions by carboxyfluorescein-diacetate succinimidyl-ester

PBMC were resuspended in PBS containing 2.5

m

M carboxyfluo-rescein-diacetate succinimidyl-ester (CFDA-SE; Molecular Probes)

404


for 10 minutes at room temperature. To quench the labeling pro-cess, an equal volume of fetal calf serum (FCS) was added. Afterwashings in PBS supplemented with 3% FCS, cells were chal-lenged with PHA (0.5

m

g/mL) for 72 hours at 37

8

C under SF condi-tions or in the presence of either preG or postG-serum, as describedearlier. CFDA-SE labeled cells then were incubated with PE-conju-gated anti-CD4 or anti-CD8 mAb or with PE-conjugated isotype-matched mAb as negative control (Caltag Laboratories) for 30 min-utes at 4

8

C. After washings with ice-cold PBS supplemented with1% BSA, cells were kept on ice until flow cytometric analysis. Cellkinetic parameters were calculated as detailed in the Appendix.

Flow cytometry

Samples were run through a FACScan flow cytometer (Becton-Dickinson [BD], Mountain View, CA, USA) equipped with an ar-gon laser emitting at 488 nm. The fluorescence of FITC andDiOC

6

(3) was recorded in FL1 (525 nm), the fluorescence of PEwas recorded in FL2 (575 nm) and the fluorescence of ethidium(Eth) was recorded in FL3 (670 nm), after suitable electronic com-pensation. A minimum of 10,000 events was acquired in list modeusing CellQuest software. Forward scatter (FSC) and side scater(SSC) were collected as linear signals, and fluorescent emissionswere collected on four-decade logarithmic scales. Optical align-ment and gain setting stability were verified daily with fluorescentmicrospheres (Calibrite; BD).

For DNA content analysis, a minimum of 30,000 events wasacquired after instrument calibration with chicken erythrocyte nu-

clei (DNA QC; BD), setting PE emission channel to a linear scalefor propidium iodide fluorescence. The calculation of cell cyclecompartments was performed by deconvoluting DNA content his-tograms on a 1,024-channel scale with ModFit LT 2.0 software(Verity Software House Inc., Topsham, ME, USA).

Statistical methods

The approximation of population distribution to normality wastested using y(g) statistics for kurtosis and symmetry. Results arepresented as mean (

m

) and standard deviation (SD). Correlationswere examined by Spearman rank analysis, and all comparisons byanalysis of variance (ANOVA) or the Student’s

t

-test for pairedand unpaired data, as appropriate. The criterion for statistical sig-nificance was defined as

p

#

0.05.

Results

Lymphocyte stimulation in the presence of postG serum is associated with collapse of

Dc

m

, hypergeneration of ROI, and caspase-3 activation

Following our observation that immunoregulatory solublefactor(s) induced by G-CSF render lymphocytes unrespon-sive to mitogenic challenge and enhance their activation-induced apoptosis [10,17], we evaluated mitochondrial in-ner transmembrane potential (

Dc

m

) and generation of ROI

Figure 1. Measurement of lymphocyte Dcm and generation of ROI after mitogenic challenge in the presence of postG serum. Normal PBMCs were chal-lenged with PHA (0.5 mg/mL) for 72 hours in the presence of preG or postG serum (20% final concentration, v/v). Control cultures were performed underserum-free (SF) conditions. (A) Lymphocyte Dcm and production of superoxide anion, as measured by DiOC6(3) retention and oxidation of HE to Eth, respec-tively, were evaluated both in cultures performed with unselected lymphocyte subsets and in those performed with FACS-purified CD41 and CD81 T cells.*p , 0.01 and **p , 0.001 compared with preG cultures. Bars represent the mean percentage of apoptotic DiOC6(3)lowEthhigh T cells from six independentexperiments performed in duplicate. (B) Representative flow cytometric profile. The percentage of apoptotic DiOC6(3)lowEthhigh CD4(8)1 T cells is indicated.The purity of FACS-selected CD41 and CD81 T-cell fractions always exceeded 99.3%.

S. Rutella et al./Experimental Hematology 29 (2001) 401–415 405

after PHA stimulation in the presence of preG or postG se-rum. To this purpose, the lipophilic cation DiOC6(3) wasused, which accumulates in the mitochondrial matrixdriven by the electrochemical gradient following theNernst equation [32]. Cells were simultaneously labeledwith hydroethidine (HE), which is oxidized by superoxideanion to Eth, emitting red fluorescence [23]. Interestingly,26% 6 7% of CD41 and 39% 6 11% of CD81 T cells inpostG cultures were apoptotic, as measured by reducedDiOC6(3) retention and hypergeneration of ROI. Con-versely, 8% 6 5% DiOC6(3)lowEthhighCD41 T cells (p ,0.01) and 6 6 3% DiOC6(3)lowEthhighCD81 T cells (p ,0.001) were found in cultures stimulated in the presence ofpreG serum (Fig. 1). When FACS-purified CD41 andCD81 T cells were challenged separately with the mitogen,the proportion of apoptotic T cells was still higher in postGcompared with preG cultures (Fig. 1), confirming that mi-tochondrial activation occurs both in unfractionated and inFACS-purified CD41 and CD81 T cells [17]. To addresswhether the perturbation of lymphocyte mitochondrialfunction is associated with caspase activation, cells werelabeled with an antibody directed against a conformationalepitope of caspase-3, an “effector” caspase responsible foramplification of the apoptotic signal and likely to be re-quired for cells to adopt a typical apoptotic phenotype [33].Of interest, active caspase-3 was detected in 45% 6 9% ofT cells from postG cultures vs 8% 6 3% of T cells in preGcultures (p , 0.001). As expected, less than 2% of T cellsreacted with the anti-active caspase-3 antibody in culturesperformed under SF conditions. The enhanced tendency toactivation-induced T-cell apoptosis in postG cultures wasconfirmed by a highly sensitive LM-PCR ladder assay(data not shown).

Lymphocyte stimulation in the presence of postG serum is associated with low expression of cyclin E and low incorporation of BrdUrdWe reasoned that the perturbation of lymphocyte Dcm andthe hypergeneration of ROI might directly impact on the ca-pacity to progress through the cell cycle; thus, the expres-sion of G1-phase cyclins was investigated and correlatedwith the cell cycle position through the bivariate analysis ofcyclin vs DNA content. Following the observation that themaximal level of D-type cyclins can be detected early aftermitogenic challenge [31], cells were harvested after 24, 48,and 72 hours of stimulation in the presence of preG orpostG serum. No difference in the overall level of D-typecyclin-associated fluorescence was found in postG vs preGcultures after 24 hours of mitogenic stimulation (Fig. 2). At48 hours of culture, the percentage of D-type cyclin2 cellswith a G0/G1-like DNA content (G0-like cells) was higher inpostG compared with preG cultures. At 72 hours of culture,the percentage of D-type cyclin1 cells with a G0/G1-likeDNA content (G1-like cells) was higher in postG comparedwith preG cultures, indicating a hindered exit from quies-cence (Fig. 2). Because the expression of D-type cyclinsprecedes that of cyclin E, the fraction of cyclin E1 lympho-cytes was measured after 72 hours of PHA stimulation. Ofinterest, an average 72% reduction of the percentage of cy-clin E1 cells was found in postG compared with preG cul-tures (Fig. 3A). A representative flow cytometric profile ofcyclin E vs DNA content is shown in Figure 3B.

When DNA synthesis was evaluated through BrdUrd in-corporation, the majority of cells stimulated in the presenceof postG serum resided in the BrdUrd2 fraction at any timeof culture. Conversely, T cells actively synthesized DNAand incorporated BrdUrd in preG cultures after 24, 48, and

Figure 2. Cell cycle position and expression of D-type cyclins after mitogenic challenge in the presence of postG serum. Normal PBMCs were activated withPHA (0.5 mg/mL) in the presence of preG or postG serum (20% final concentration, v/v). Control cultures were performed under serum-free (SF) conditions.T cells were gated based on CD3 specific fluorescence. D-type cyclin2 T cells with a G0/G1 DNA content were considered to be in the G0 phase of the cellcycle. D-type cyclin1 T cells with a G0/G1 DNA content were considered to be in the G1 phase of the cell cycle. Cells were considered in the G2-M phase ofthe cell cycle if they expressed a G2-M DNA content and they were positive for D-type cyclins. *p , 0.01 and **p , 0.001 compared with preG cultures.Datapoints represent the mean and standard deviation obtained in six independent experiments performed in duplicate.

406 S. Rutella et al./Experimental Hematology 29 (2001) 401–415

72 hours of mitogenic challenge, and reacted positively withthe anti-BrdUrd mAb (Fig. 4A). However, the percentage ofBrdUrd1 T cells was higher in cultures performed under SFconditions compared with preG cultures. These findingswere not unexpected, because the supplementation of cul-ture medium with serum might affect negatively cell expan-sion [34]. A representative flow cytometric profile is shownin Figure 4B.

Cell division tracking of lymphocytesstimulated in the presence of postG serumThe percentage of undivided CD41 and CD81 T cells wassignificantly higher in postG compared with preG cultures.Whereas up to four cell generations of progeny could beidentified in preG cultures, the majority of cells challengedwith PHA in the presence of postG serum underwent tworeplications (Fig. 5A). A flow cytometric profile of CFDA-SE fluorescence is depicted in Figure 5B. The proliferationindex, as defined in the Appendix, was significantly lowerin postG compared with preG cultures (1.44 6 0.4 vs 1.83 60.5; p 5 0.02) and with cultures performed under SF condi-tions (2.42 6 0.7; p , 0.01; Fig. 5C). As expected, the re-covery of label at time t 5 72 hours, i.e., a function of theratio of expansion to proliferation, was higher in postGcompared with preG cultures (33% 6 8% vs 17% 6 7%;p 5 0.001; Fig. 5C). When the probability of undividedcells to undergo i divisions at time t 5 72 hours was calcu-

lated, this parameter was significantly higher in preG com-pared with postG cultures in the first three generations ofprogeny. No differences in the probability of cell divisionwere found in CD41 vs CD81 T cells from postG cultures,suggesting that the soluble activity contained in postG se-rum equally affected the proliferative response of CD41 aswell as CD81 T cells (Fig. 5D).

Lymphocytes en route toapoptosis express hyperphosphorylated pRBGiven the pivotal role of the pocket proteins pRB and p107in the advancement through the cell cycle, we investigatedtheir expression and phosphorylation state by Western blot-ting. After 72 hours of mitogenic stimulation, the cellularproteins were probed with antibodies that recognize boththe hypophosphorylated (G1-phase) and the hyperphospho-rylated (S-phase) form of pRB and p107. PHA activationwas associated with upregulation of both pRB and p107 lev-els compared with the unstimulated control (Fig. 6A). Sur-prisingly, a slow-migrating hyperphosphorylated (S-phase)pRB and p107 band was evident even in growth-arrestedpostG cultures (Fig. 6B). Similar results were obtained fol-lowing shorter incubation times with the mitogen (data notshown). No statistically significant differences in either thepercentage of pRB1 and p1071 T cells or the mean fluores-cence intensity of the antigen-positive T-cell populationwere detected by flow cytometric analysis, indicating that

Figure 3. Expression of cyclin E after mitogenic challenge in the presence of postG serum. Normal PBMCs were challenged with PHA (0.5 mg/mL) in thepresence of preG or postG serum (20% final concentration, v/v). Control cultures were performed under serum-free (SF) conditions. T cells were gated basedon CD3 specific fluorescence. (A) Mean and standard deviation obtained in six independent experiments performed in duplicate. An average of 35% 6 7% ofT cells reacted positively with the anti–cyclin-E mAb in preG cultures, whereas cyclin-E expression was detected on 10% 6 3% of T cells in postG cultures.*p , 0.001. (B) Representative flow cytometric profile of cyclin-E expression vs DNA content. Markers were set according to the proper isotypic control, andthe percentage of cyclin-E1 T cells is indicated.


the overall quantity of pRB and p107 was similar in postGvs preG cultures (Fig. 6C).

Lack of CDKI recruitment in growth-arrested lymphocytesThe expression pattern of representative members of theINK4 and Cip/Kip families of CDKI was investigated next.Quiescent lymphocytes expressed p15INK4B, albeit at low lev-els, as previously reported [35]. PHA stimulation was associ-ated with the increase of p15INK4B specific fluorescence. Un-expectedly, 20% 6 8% of T cells in postG culturesexpressed p15INK4B compared with 50% 6 14% of T cellsactivated in the presence of preG serum (p , 0.01; Fig. 7A).When p16INK4A expression was assayed, a similar patternwas found: 37% 6 12% of T cells in postG cultures wererecognized by the anti-p16INK4A antibody compared with69% 6 18% in preG cultures (p 5 0.001; Fig. 7A). Further-more, the expression levels of p27Kip1 were lower in postGcompared with preG cultures (46% 6 15% vs 65% 6 20%;p , 0.001), whereas no modification of p21Cip1 levels wasfound, with more than 90% of T cells reacting positivelywith the anti-p21Cip1 mAb in any culture condition (Fig. 7A).

In line with our observation of higher BrdUrd incorpora-tion under SF conditions, p21Cip1 was expressed at lower in-tensity in SF cultures (mean fluorescence intensity 185 645 arbitrary units) compared with preG cultures (mean fluo-rescence intensity 260 6 50 arbitrary units; p , 0.05). Sim-

ilarly, p27Kip1 levels were lower in SF cultures comparedwith preG ones. Whereas in the former p27Kip1 was ex-pressed by 40% 6 15% of the cells, albeit at low intensity,in the latter a biphasic staining pattern was recognized and70% 6 8% of the cells expressed p27Kip1 at intermediatelevels (Fig. 7A). These phenomena were anticipated andconceivably reflected an inverse relationship betweenp21Cip1 or p27Kip1 levels and cell proliferation rate both inSF and in preG cultures. The unanticipated finding was theunscheduled expression of p21Cip1 or p27Kip1 in postG cul-tures, where inhibited cell cycle traverse was associatedwith lack of p21Cip1 or p27Kip1 recruitment. Interestingly, nosignificant changes of p15INK4B and p16INK4A staining inten-sity were found when comparing SF and preG cultures. Col-lectively, it can be stated that p15INK4B, p16INK4A, andp27Kip1 levels were reduced by 50%, 70%, and 40% inpostG vs preG cultures (Fig. 7B).

N-acetylcysteine andamifostine can inhibit activation-inducedapoptosis and restore cell cycle progressionWe tested the ability of antioxidant and cytoprotectant agentsto prevent the G-CSF–induced mitochondrial dysfunction. Toaddress this point, lymphocytes were preincubated with NAC(10 mM/L) or amifostine (60 mg/mL) for 30 minutes prior toPHA stimulation in the presence of preG or postG serum. Be-

Figure 4. BrdUrd incorporation after mitogenic challenge in the presence of postG serum. Normal PBMCs were challenged with PHA (0.5 mg/mL) for 72hours in the presence of preG or postG serum (20% final concentration, v/v). Control cultures were performed under serum-free (SF) conditions. (A) BrdUrdincorporation after 24, 48, and 72-hour PHA-activation. Mean and standard deviation observed in five independent experiments performed in duplicate. *p ,0.01 and **p , 0.001 compared with preG cultures. (B) Representative flow cytometric profiles. Markers were set according to the proper isotypic control.The percentage of cells that incorporate BrdUrd as a result of DNA synthesis (S-phase cells) and react with the anti-BrdUrd mAb is indicated.


cause of its powerful survival-enhancing effects, IL-2 wasused at 10 IU/mL to obtain a positive control for inhibited ap-optosis. Pretreatment with both NAC and amifostine was as-sociated with an average 45% reduction of apoptotic cell

death of CD41 as well as CD81 T cells from postG cultures(Fig. 8A). As expected, IL-2 was capable of further antago-nizing activation-induced cell death, which was reduced by60% on average (data not shown). Of interest, preincubation

Figure 5. Cell division tracking after mitogenic challenge in the presence of postG serum. Normal PBMCs were first loaded with CFDA-SE (2.5 mM) for 10minutes and then challenged with PHA (0.5 mg/mL) for 72 hours in the presence of preG or postG serum (20% final concentration, v/v). Control cultures wereperformed under serum-free (SF) conditions. The analysis of CFDA-SE fluorescence profiles was performed with the proliferation wizard of the ModFitLT 2.0 software (Verity Software House Inc.), following the assumption that CFDA-SE intensity at each generation exhibits a Gaussian distribution. SeeAppendix for details. (A) Percentage of undivided (parental) cells and of cells residing in the first five generations of progeny as determined by CFDA-SE flu-orescence. Mean and standard deviation recorded in six independent experiments performed in duplicate. T cells were gated and analyzed based on CD4 andCD8 specific fluorescence. *p , 0.01 compared with preG cultures. (B) Representative flow cytometric profiles of CFDA-SE fluorescence after PHA-stimu-lation in the presence of preG or postG serum. The histograms obtained after 24 hours of culture were used to establish the position of undivided (parental)cells (peak 0). Peaks 1 through 8 were assigned based on the decrease in CFDA-SE fluorescence per cell (two-fold loss per generation). See Appendix for fur-ther details. T cells were gated and analyzed based on CD4 and CD8 specific fluorescence. (C) Probability to undergo i divisions for the first five generationsof progeny after PHA challenge in the presence of preG or postG serum. Mean and standard deviation observed in six independent experiments performed induplicate are plotted on the y-axis. See Appendix for details. *p , 0.01 compared with preG cultures. (D) Proliferation index (PI) and label recovery (rt) incultures performed under SF conditions or in the presence of preG or postG serum. The PI of unstimulated cultures was 1.2 6 0.6 (right panel). Mean andstandard deviation observed in six independent experiments performed in duplicate are plotted on the y-axis. See Appendix for details. *p , 0.01 comparedwith preG cultures.


with both NAC and amifostine strongly inhibited the activa-tion and processing of caspase-3 in postG cultures (Fig. 8B).

When the effects of NAC and amifostine on cell cycleprogression were evaluated, an average 3.5-fold increase ofBrdUrd incorporation was found in postG cultures preincu-bated with NAC or amifostine, suggesting that the protec-tion of cell mitochondria was paralleled by the restorationof DNA synthesis (Fig. 8C). Similarly, cyclin-E levels werecomparable to those observed in preG cultures (data notshown). Taken together, these findings indicate that mito-chondrial dysfunction was likely to account for the hinderedcell cycle traverse and that the prevention of apoptotic celldeath through the maintenance of lymphocyte Dcm can fa-vorably impact on cell cycle progression.

DiscussionG-CSF, a growth and differentiation factor for neutrophils[36], can be used successfully to mobilize hematopoieticprogenitors from healthy donors for transplantation into al-logeneic recipients [1–6]. Several explanations have beenproposed for the unexpectedly reduced incidence and sever-ity of acute GVHD following PBPC compared with bonemarrow allografting. In particular, G-CSF exerts powerfulanti-inflammatory effects, consisting of attenuation of Th1cytokine release and polarization of T-cell responses towarda Th2 cytokine profile [9,37–39]. We and others demonstratedthat G-CSF–induced immunoregulatory mediators limit T-cellproliferation and enhance activation-induced T-cell apoptosis[9,10,17]; however, the molecular mechanisms underlying

Figure 6. Western blot and immunofluorescence analysis of pRB and p107 expression after mitogenic challenge in the presence of postG serum. NormalPBMCs were challenged with PHA (0.5 mg/mL) for 72 hours in the presence of preG or postG serum (20% final concentration, v/v). Control cultures wereperformed under serum-free (SF) conditions. (A) Western blot analysis of pRB and p107 phosphorylation state after 72-hour PHA activation. The blots wereincubated with polyclonal antibodies directed against p107 and pRB. Detection was performed by chemiluminescence. The molecular weight standardincluded the 115-kDa band used as a reference; its position is indicated on the left. The fast-migrating hypophosphorylated (hypo-PO4) and the slow-migratinghyperphosphorylated (hyper-PO4) forms are indicated on the right. Lane 1 5 unstimulated cells; lane 2 5 cells stimulated with PHA under serum-free (SF)conditions; lane 3 5 cells stimulated with PHA in the presence of preG serum; lane 4 5 cells stimulated with PHA in the presence of postG serum. Note thatboth the hypophosphorylated (G1 phase) and the hyperphosphorylated (S phase) pRB bands appear in postG cultures. Similar results were obtained in sixindependent experiments. (B) Image analysis and quantification of pRB and p107 immunoblots (see Materials and methods for details). Band intensity wasexpressed as fold-increase of relative absorbance units in PHA-challenged compared with unstimulated cultures. 2 5 Cells stimulated with PHA under serum-free (SF) conditions; 3 5 cells stimulated with PHA in the presence of preG serum; 4 5 cells stimulated with PHA in the presence of postG serum. Mean andstandard deviation recorded in six independent experiments are shown. (C) Overall levels of pRB and p107 protein as measured by flow cytometry after chal-lenge with PHA in the presence of preG or postG serum. The marker was set according to the proper isotypic control. The percentage of cells positive for eachantigen is indicated for this representative experiment.


these phenomena have not been thoroughly investigated[16,24].

The regulation of cell cycle progression by the cyclinsand CDKs has been extensively characterized [40–44]. Re-cent experimental evidence supports the view that cell cycleand apoptosis are interconnected and that apoptosis regula-tory proteins directly impinge on the cell cycle machinery.Consistent with this model, the transition from the G1 to theS phase of the cell cycle represents a putative “apoptosis re-striction point,” where cells would be highly susceptible toimplement a death program [45–47]. Furthermore, mito-chondrial dysfunction might contribute to the initiation ofapoptotic events. In this regard, loss of Dcm, a process re-ferred to as permeability transition, might lead to enhancedgeneration of superoxide anion, release of apoptosis-induc-ing-factor and cytochrome c, and, eventually activation ofapoptogenic caspases [48].

Although limited information is available on the modula-tion of components of the cell cycle machinery in responseto the perturbation of mitochondrial function, it has been re-ported that the arrest in G1 phase of the cell cycle can occurirrespective of the recruitment of CDKI, suggesting that, at

least in some experimental models, alternative pathwaysregulated at the level of cellular energetic checkpointsmight operate to hinder cell cycle progression [47].

The present study was aimed at evaluating the expressionof key components of the cell cycle machinery and to corre-late it with the increased tendency to activation-inducedT-cell apoptosis after the exposure to postG serum [17]. Inaccordance with previously published data [17], CD41 andCD81 T cells challenged in the presence of postG serumdisplayed signs of mitochondrial activation, i.e., reducedDiOC6(3) retention and hypergeneration of ROI. These phe-nomena were associated with the activation and processingof caspase-3 and with fragmentation of genomic DNA. Pre-incubation with the antioxidant and cytoprotectant agentsNAC and amifostine, known to favor the maintenance ofDcm [49], was associated with inhibition of caspase-3 acti-vation/processing and reduction of lymphocyte apoptosis,which, however, could not be prevented solely by protect-ing cell mitochondria.

We then evaluated the expression of G1-phase cyclins af-ter 72-hour PHA stimulation in the presence of postG se-rum. We found that cells resided predominantly in a G1-like

Figure 7. Analysis of CDKI expression after mitogenic challenge in the presence of postG serum. Normal PBMCs were challenged with PHA (0.5 mg/mL) for72 hours in the presence of preG or postG serum (20% final concentration, v/v). Control cultures were performed under serum-free (SF) conditions. T cells weregated based on CD3 specific fluorescence. (A) Representative flow cytometric profiles of CDKI expression. The marker was set according to the proper isotypiccontrol. The percentage of cells positive for a given antigen is indicated. (B) Relative change in p15INK4B, p16INK4A, p21Cip1, and p27Kip1 expression levels inpostG compared with preG cultures. Mean and standard deviation recorded in six independent experiments performed in duplicate are plotted on the y-axis.


phase of the cell cycle, as measured by the combined analy-sis of D-type cyclins and DNA content. Furthermore, a 72%average reduction of cyclin-E levels occurred in postG com-pared with preG cultures. These phenomena were associ-ated with a significant reduction of BrdUrd incorporation inpostG cells both at 48 and 72 hours of culture. Taken to-gether, these data suggest that exit from quiescence and cellcycle progression were hindered by soluble factor(s) con-tained in postG serum. Furthermore, both NAC and amifos-tine were capable of partially restoring DNA synthesis andBrdUrd incorporation in postG cultures, indicating an inter-connection between mitochondrial dysfunction and inhib-ited advancement through the cell cycle in our experimentalmodel.

The expression of representative members of the INK4family of CDKI, namely, p15INK4B and p16INK4A, was signif-icantly lower in postG compared with preG cultures. Thesefindings were surprising, because CDKI levels were ex-pected to increase in growth-arrested cells. However, bothp16INK4A and p15INK4B accumulate in senescent cells [50];thus, higher levels of the INK4 proteins might reflect ahigher number of populations doublings in preG comparedwith postG cultures. This hypothesis is supported by celltracking experiments that demonstrated a lower number ofcell generations and a lower probability to undergo cell di-vision in postG compared with preG cultures. Furthermore,it is noteworthy that p27Kip1 levels were lower after chal-lenge in the presence of postG compared with preG serum.The known biologic functions of p27Kip1 include mainte-nance of cells in quiescence, control of cell cycle restrictionpoint, and promotion of cell cycle exit in response to antim-itogenic stimula and differentiation [51,52]. In view of evi-dence that p27Kip1 can prevent cell death induced by growthfactor withdrawal and safeguard against apoptosis understress conditions [53], it can be hypothesized that the re-duced levels of p27Kip1 observed in postG cultures contrib-uted to the enhanced susceptibility to apoptosis. As an addi-tional mechanism, the reduction of p27Kip1 might beascribed to Bax protein overexpression, which has beenshown in lymphocytes exposed to postG serum and whichmight accelerate p27Kip1 degradation [17,45].

The induction of p21Cip1 in response to various stimuliresults in inhibition of G1cyclin/CDK complexes and G1 ar-rest, which can be related to pRB dephosphorylation and in-activation of E2F transcription factor [54]. In our study,lymphocyte activation in the presence of postG serum wasnot accompanied by a dysregulated p21Cip1 induction. Basedon the present results, it seems plausible that individual

Figure 8. Effect of antioxidant agents on lymphocyte apoptosis andcycling after mitogenic challenge in the presence of postG serum. NormalPBMCs were preincubated with NAC (10 mM/L) or amifostine (60 mg/mL)for 30 minutes prior to 72-hour PHA stimulation in the presence of eitherpreG or postG serum. (A) CD41 and CD81 T-cell apoptosis was evaluatedwith the fluorescent probes DiOC6(3) and HE, as described previously. Thepercentage of apoptotic T cells is indicated on the y-axis. Bars representmean and standard deviation recorded in six independent experiments per-formed in duplicate. *p , 0.01 compared with postG cultures not preincu-bated with NAC or amifostine. (B) T cells were gated based on CD3 spe-cific fluorescence, and active caspase-3 levels were measured with apolyclonal rabbit antibody reacting with a conformational epitope exposedon the processed forms of caspase 3. Bars represent mean and standarddeviation recorded in six independent experiments performed in duplicate.*p , 0.01 compared with postG cultures not preincubated with NAC oramifostine. (C) T cells were gated based on CD3 specific fluorescence.

Bars represent mean and standard deviation observed in six separate exper-iments performed in duplicate. An average 3.5-fold increase in the percent-age of BrdUrd1 T cells was found in postG cultures as a result of DNAsynthesis. *p , 0.01 compared with postG cultures not preincubated withNAC or amifostine.


stimuli elicit the participation of a specific CDKI for induc-tion of the G1 arrest response. Of interest, a different andspecific role of p21Cip1 and p27Kip1 was demonstrated inother system models. In particular, p21Cip1 expression mightbe related to proliferation of myeloid precursor cells, declin-ing during terminal differentiation, whereas p27Kip1 levelsremain fairly constant [55,56].

pRB expression is crucial for the commitment of cells toenter S phase and is upregulated during the G1-phase. Innormal cells, pRB appears as a hypophosphorylated 110-kDa species, whereas at the G1–S boundary pRB undergoesprogressive phosphorylation by the cyclin–CDK com-plexes, resulting in a 144-kDa form [57]. Interestingly, adual role for pRB in the inhibition of cellular proliferationhas been described, namely, induction of cell cycle arrestand promotion of cell death [22,57–59]. In this respect,abrupt pRB phosphorylation might result in loss of functionas a coordinator of the G1–S transition and might trigger celldeath. Moreover, phosphorylation at particular sites of pRBmight be sufficient for apoptosis induction [60]. An interest-ing model of apoptotic cell death has been described in den-sity-arrested murine 3T3 fibroblasts, which, upon releasefrom quiescence, upregulate early cell cycle genes, phos-phorylate pRB, and reach the G1–S border en route to apop-tosis; thus, cells might exhibit molecular events mimickingthe G1 phase transition but leading to apoptotic cell deathrather than replication [61]. An unanticipated and intriguingfinding of this study was that pRB and p107 were both inthe hypophosphorylated and the hyperphosphorylated formsin growth-arrested T cells from postG cultures; however,the overall quantity of pRB and p107 was unchanged com-pared with preG cultures, as shown by image analysis of theimmunoblots and by flow cytometry.

In conclusion, this study provides, for the first time, aninsight into the molecular mechanisms associated with theG-CSF–induced T-cell mitogenic unresponsiveness. Al-though the phosphorylation of pRB and the downregulationof CDKI imply that cells have become competent for entryinto the S phase of the cell cycle upon mitogenic activation,soluble factor(s) elicited by G-CSF hinder the passagethrough the G1 restriction point, favoring the execution ofan apoptotic program (Fig. 9).

Transforming growth factor-b1 (TGF-b1) is an intrigu-ing regulator of immune homeostasis, and its presence dur-ing T-cell stimulation is associated with enhanced T-cell ef-fector potential, increased resistance to apoptosis, long-termsurvival, and memory generation [62–64]. Interestingly,TGF-b1 null T cells are characterized by dissipation of Dcm

and enhanced susceptibility to apoptosis, strongly indicatingthat TGF-b1 might dissociate death receptor signaling fromdownstream effector pathways [65]. Although no signifi-cant fluctuations of serum TGF-b1 over the threshold levelcould be detected by ELISA in healthy PBPC donors treatedwith G-CSF (personal unpublished observation), it is tempt-ing to speculate that reduced levels of endogenous/autocrineTGF-b1 in postG cultures might inhibit lymphocyte prolif-eration and enforce apoptosis. Preliminary observationssuggest a downregulation of autocrine TGF-b1 both atmRNA and protein levels in lymphocytes activated in thepresence of postG serum (manuscript in preparation). Thesefindings offer an additional explanation for the reportedlack of p15INK4B and p27Kip1 induction in postG cultures.The role of TGF-b1 in the G-CSF–induced immune sup-pression is under active investigation.

Whether G-CSF can be beneficial as an immunomodula-tor in acute GVHD and autoimmune diseases remains to be

Figure 9. Overview of G-CSF–induced immune dysfunction. In this model, it is proposed that soluble factors contained in postG serum hinder cell cycle pro-gression, inducing a molecular phenotype that mimicks the G1–S phase traverse and consists of pRB phosphorylation, lack of CDKI recruitment, and reducedcyclin-E expression. These features are associated with a profound perturbation of mitochondrial function, with Bax protein overexpression and caspase-3activation/processing, ultimately leading to DNA fragmentation and apoptosis. Recent studies have suggested the involvement of the CD95-CD95L pathwayin G-CSF–induced apoptotic cell death. Thus, the putative relationship between lymphocyte mitogenic unresponsiveness and apoptosis induction would occurat the level of key molecules shared by the cell cycle and apoptotic machineries. The immunoregulatory soluble factor(s) responsible for these phenomenahave yet to be identified. See text for details and references.


evaluated [66]. In particular, it will be necessary to assess theclinical translation of these basic observations in patientstransplanted with G-CSF–mobilized allogeneic PBPC.

Appendix

The analysis of CFDA-SE fluorescence profiles was per-formed with the proliferation wizard of the ModFit LT 2.0software (Verity Software House Inc.). Following the as-sumption that CFDA-SE intensity at each generation exhib-its a Gaussian distribution, the reduction of CFDA-SE fluo-rescence by 50% in PHA-challenged compared with controllymphocytes indicated one cell division and further reduc-tions by 50% indicated each additional cell division. Spac-ing between generations was 19.19 channels on a four-decade logarithmic scale. Replication data were expressedin terms of “proliferation index” (PI), which was calculatedaccording to Equation 1 [67]:

(1)

where G represents the number of events in a given genera-tion and n the generation number (range 1–8). The PI of apopulation not containing cells that underwent division is1.0; when all cells have divided once, the PI value is 2.0; in-creasing numbers of replications are reflected by higher PIvalues. The probability of an undivided cell to undergo i di-visions, pi(t), has been defined as given in Equation 2:

(2)

where the number of undivided cells is calculated from thenumber of cells in a given generation by dividing by 2i [68].The expansion («t) provides the net increase in cell numberat time t compared with the inoculum number at time t 5 0and has been calculated as given in Equation 3:

(3)

The total number of undivided cells estimates viable parentalcells at time t. If a proportion of the inoculum does not sur-vive, this estimate will be less than the number of cells at t 50. The proliferation, wt, was defined as given in Equation 4:

(4)

The ratio of expansion to proliferation provides an esti-mate of CFDA-SE recovery (rt) and cell survival at time t,and has been calculated as given in Equation 5:

(5)

PIGn

n 1=∑

Gn2n 1–---------------

n 1=∑-----------------------=

pi t( )

Number of undivided cells giving rise to i generationsTotal number of undivided cells

---------------------------------------------------------------------------------------------------------------------------------

=

εtTotal number of cells at time tTotal number of cells at t 0=------------------------------------------------------------------------=

ϕ tTotal number of cells at time t

Total number of undivided cells-----------------------------------------------------------------------------=

ρtεtϕ t----- 1≤=

AcknowledgmentsThis work was supported by a grant from Associazione Italiana perla Ricerca sul Cancro (AIRC).

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T-cell apoptosis induced by granulocyte colony-stimulating factor is associated with retinoblastoma...

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