IMMUNOBIOLOGY
Reduced frequencies and suppressive function of CD4ϩCD25hi regulatory T cells in patients with chronic lymphocytic leukemia after therapy withfludarabine
Marc Beyer,Matthias Kochanek,Kamruz Darabi,Alexey Popov,Markus Jenn,Elmar Endl,Percy A.Knolle,Roman K.Thomas, Michael von Bergwelt-Baildon,Svenja Debey,Michael Hallek,and Joachim L.Schultze
Globally suppresd T-cell function has been described in many patients with cancer to be a major hurdle for the devel-opment of clinically efficient cancer immu-notherapy.Inhibition of antitumor im-mune respons has been mainly linked to inhibitory factors prent in cancer patients.More recently,incread fre-quencies of CD4؉CD25hi regulatory T cells (T reg cells)have been described as an additional mechanism reducing immu-nity.We assd73patients with B-cell chronic lymphocytic leukemia(CLL)and 42healthy controls and demonstrated
significantly incread frequencies of cy-
totoxic T lymphocyte-associated protein
4(CTLA4؉)–,Forkhead box P3(FOXP3؉)–,
glucocorticoid-induced tumor necrosis
insistencefactor receptor-related protein(GITR؉)–,
CD62L؉–,transforming growth factor1
(TGF-1؉)–,interleukin10(IL-10؉)–T reg
cells in patients with CLL,with highest记承天寺夜游原文及翻译
frequencies in untreated or progressing
patients prenting with extended dis-
ea.Most surprisingly,in the majority of
patients with CLL treated withfludarabine-
containing therapy regimens the inhibi-
tory function of T reg cells was decread
or even abrogated.In addition,frequen-
新年快乐英文cies of T reg cells were significantly de-
领略什么cread after therapy withfludarabine.In
light of similarfindings for cyclophospha-
mide the combination offludarabine and
cyclophosphamide might be further ex-
ploited in strategies reducing immunosup-
pression prior to cancer immunotherapy.
(Blood.2005;106:2018-2025)
©2005by The American Society of Hematology
Introduction
Human and murine CD4ϩCD25ϩT cells contain cells that suppress antigen-specific T-cell immune respons.1-5The naturally occur-ring regulatory CD4ϩCD25ϩT cells originate from the thymus and play a central role in the maintenance of peripheral tolerance by suppression of autoreactive T-cell populations.In murine models, regulatory T cells(T reg cells)prevent autoimmune and inflamma-tory dias1,6,7and inhibit antitumor immune respons.8-12 Although a truly unique marker for T reg cells is still not available, veral molecules have been associated with the cells including cytotoxic T lymphocyte-associated protein4(CTLA4),13-16glu-cocorticoid-induced tumor necrosis factor receptor-related protein (GITR,TNFRSF18),17,18Forkhead box P3(FOXP3),19-21L-lectin(CD62L,SELL),22,23and OX40antigen(CD134, TNFRSF4).23,24
In humans,T reg cells are enriched within the CD4ϩCD25hi population,whereas CD4ϩCD25lo T cells reprent mainly previ-ously activated T helper cells.25The CD4ϩCD25hi T reg cells inhibit proliferation and cytokine relea by conventional CD4ϩCD25ϪT cells.26Decrea of the cells was found in patients with autoimmune dias,27-31whereas an increa of T reg cells in patients after allogeneic bone marrow transplantation was associated with a reduced graft-versus-host dia.32-35In patients with malignant melanoma,36Hodgkin lymphoma,37or ovarian,38,39gastric,
40,41lung,39,42breast,43,44and pancreatic cancer43inhibitory CD4ϩCD25ϩT cells are also incread.In an elegant study,Curiel et al38demonstrated that functional T reg cells were enriched in ascites from women with ovarian cancer,migrated toward CCL22 expresd by tumor cells and tumor-associated macrophages,and specifically inhibited antitumor immunity.Moreover,within this tting,the increa of T reg cells predicted poor survival.38Only recently,studies asssing a potential influence of chemotherapy on T reg cells have been initiated.In mice,low-do cyclophosphamide decread the number of T reg cells.45
Bad on the obrvations we were interested in understand-ing whether CD4ϩCD25hi T cells are also incread and posss inhibitory capacities in B-cell chronic lymphocytic leukemia (CLL)and,if so,to asss the frequency and function in the context of stage of dia and prior therapy.CLL,the most common type of leukemia in the Western hemisphere,46is characterized by clonal proliferation and accumulation of neoplastic B lymphocytes.47-49 CLL is a particularly interesting model becau it is frequently associated with clinically manifest immune defects,suggesting an underlying immune dysregulation.50-56In fact,decread T-cell respons to mitogenic and T-cell receptor-mediated stimulations have been described in patients with CLL57,58;however,the accounting cellular and molecular mechanisms are still unclear.59
From the Molecular Tumor Biology and Tumor Immunology,University of Cologne,Cologne,Germany;Clinic I for Internal Medicine,University of Cologne,Cologne,Germany;and Institute for Molecular Medicine and Experimental Immunology,University of Bonn,Bonn,Germany.
Submitted February15,2005;accepted May15,2005.Prepublished online as Blood First Edition Paper,May24,2005;DOI10.1182/blood-2005-02-0642. Supported by the Sofja Kovalevskaja Award of the Alexander von Humboldt-Foundation(J.L.S.)and the Wilhelm-Sander Stiftung(J.L.S.and M.K.).M.B.B. is supported by a Carreras Foundation Fellowship and a Max Eder Award from the Deutsche Krebshilfe.P.A.K.and E.E.are supported in part by grant HBFG-109-517.M.B.and ibuted equally to this work.
The online version of the article contains a data supplement.
Reprints:Joachim L.Schultze,Molecular Tumor Biology and Tumor Immunology Clinic I for Internal Medicine,University of Cologne,Joph-Stelzmann Str 9/Haus16,50931Cologne,Germany;e-mail:joachim.schultze@uk-koeln.de.
The publication costs of this article were defrayed in part by page charge payment.Therefore,and sol
ely to indicate this fact,this article is hereby marked‘‘advertiment’’in accordance with18U.ion1734.
©2005by The American Society of Hematology
2018BLOOD,15SEPTEMBER2005⅐VOLUME106,NUMBER6
Moreover,chemotherapy applied to patients with CLL includes drugs such asfludarabine,cyclophosphamide,or alemtuzumab, which are cytotoxic for T cells and have been shown to alter ratios of CD4ϩto CD8ϩT cells in vitro.60
Overall,we assd T reg cells in73patients with CLL and42 healthy individuals.In addition to prenting clear evidence of a stage-dependent increa of T reg cells in this leukemia,we ob-rved,for thefirst time in humans,a significant impact of chemotherapy,particularlyfludarabine-bad therapy regimens,on the frequency and function of CD4ϩCD25hi T cells. Patients,materials,and methods
Patients and clinical parameters2014安徽高考英语
Peripheral blood from42healthy individuals and73patients with CLL was obtained following approval
by our institutional review board(University Ethics Committee,Cologne,Germany),including35patients in whom we
assd blood samples at least at2different time points.Informed connt for blood donations was obtained,per the Declaration of Helsinki,from all volunteers.Peripheral blood mononuclear cells(PBMCs)from healthy donors(controls)and patients meeting diagnostic criteria for CLL were obtained from peripheral blood using Ficoll/Hypaque(Amersham,Uppsala, Sweden)density centrifugation and stored in liquid nitrogen until further u.Patients included for phenotypical or functional analysis were either untreated or had not received cytoreductive treatment for a period of at least 1month before investigation.Staging was performed according to the Binet classification for CLL.The mean age of the patients with CLL atfirst analysis was61.2Ϯ10.4years and that for the corresponding healthy controls,43.0Ϯ14.0years.Clinical characteristics of the patients studied are summarized in Table S1,available on the Blood website(e the Supplemental Materials link at the top of the online article).Variance analysis to asss dependency of age and frequency of CD4ϩCD25hi T cells in healthy donors and patients with CLL did not reveal a correlation between the variables.
Antibodies andfluorescence-activated cell sorting analysis Cell phenotype of T cells within PBMCs w
as defined by multicolorflow cytometry using the following antibodies:fluorescein isothiocyanate(FITC)–conjugated CD4;phycoerythrin(PE)–conjugated CTLA4(CD152);PE-cyanin5(Cy-5)–conjugated CD25,–CD45RA,and–CTLA4;allophycocya-nin(APC)–conjugated CD4;APC-Cy-7–conjugated CD4(all from Becton Dickinson[BD]PharMingen,Heidelberg,Germany);PE-conjugated CD25, CD62L;peridinin chlorophyll protein(PerCP)–conjugated CD3;PE-Cy-7–conjugated CD25(all from Becton Dickinson Biosciences,Heidelberg, Germany);and the corresponding isotype control antibodies(BD PharMin-gen).Cells were stained according to the manufacturer’s recommendations. For intracellular staining cells were permeabilized using Cytofix/Cytoperm solution(BD PharMingen)after surface staining and incubated with interleukin10(IL-10)–FITC,GITR-FITC(both from R&D Systems, Wiesbaden,Germany),transforming growth factor1(TGF-1)–PE (IQ-Products,Groningen,The Netherlands),and CTLA4-PE(BD PharMin-gen)or with the appropriate isotype controls(BD PharMingen).Samples were then washed and stored at4°C until acquisition.
Samples were acquired either on a FACSCalibur or FACSCanto(both from BD Biosciences)and analyzed with CELLQuest or FACSDiva software(both from BD Biosciences)or WinMDI2.8(facs.scripps.edu/ software.html).CD25lo and CD25hi T cells were gated as demons
trated in Figure1A for all samples analyzed according to previously published data.25The analysis was performed independently by2investigators(M.B. and J.L.S.)with similar results.Frequencies of CD4ϩCD25hi T cells in peripheral blood are shown as percent values of CD4ϩT cells.To determine cells positive for additional T reg cell markers,we ud stringent gating criteria,tting gates at the1%level of the respective isotype control.CD4؉T-cell isolation and culture
CD4ϩT cells were purified from PBMCs using CD4magnetic-activated cell sorting(MACS)beads(Miltenyi Biotec,Bergisch Gladbach,Germany) as described previously.61To asss polyclonal CD4ϩT-cell activation 1ϫ105CD4ϩT cells/well were activated in AIM-V(Gibco Invitrogen, Karlsruhe,Germany)/EX-Cell610(JRH Biosciences,Lenexa,KS)with anti-CD3(0.2g/mL,OKT-3)and anti-CD28monoclonal antibody(mAb;
0.2g/mL,kind gift of Dr L.M.Nadler,Dana-Faber Cancer Institute, Boston,MA)in96-well plates.The proliferation of T cells was monitored by measuring incorporation of5-bromo-2Ј-deoxyuridine(BrdU;Roche Diagnostics,Mannheim,Germany)on day2to3of culture.Cells were harvested24hours after the addition of BrdU.BrdU incorporation was assd by absorbance at a wavelength of450nm using a multiwell enzyme-linked immunosorbent assay(ELISA)reader.
Isolation of CD4؉CD25hi and CD4؉CD25؊T cells
In18patients with CLL we were able to obtain sufficient amounts of peripheral blood(Ͼ50mL)to isolate CD4ϩCD25ϪT cells and CD4ϩCD25hi T cells from PBMCs for functional analysis.14Briefly,CD4MACS multisort beads(Miltenyi Biotec)were ud for isolation of CD4ϩT cells. After detaching,cells were washed and CD4ϩCD25hi T cells were positively lected using CD25microbeads(2L beads/107CD4ϩT cells). The described technique is optimized for the isolation of human CD4ϩCD25hi T cells with high purity.62U of higher concentrations of microbeads for isolation results in better recovery but decreas the purity.The negative fraction of CD4ϩCD25ϪT cells was ud as effectors.Alternatively,CD4ϩT cells were isolated with CD4MACS beads as described earlier and stained with CD4-FITC and CD25-PE.CD4ϩCD25hi T cells were purified using a FACSDiVa cell sorter(BD Biosciences).Cells were reanalyzed after sorting and routinely showed more than95%purity.For some experiments,CD4ϩCD25hi as well as CD4ϩCD25ϪT cells were preacti-vated with0.5g/mL anti-CD3mAb at37°C for20hours in the prence of10U/mL IL-2(Proleukin;Chiron,Munich,Germany)in X-VIVO15 (BioWhittakker,Verviers,Belgium).
RNA extraction and real-time rever transcription–PCR
for FOXP3
Total RNA from purified CD4ϩCD25Ϫand CD4ϩCD25hi T cells was isolated using TRIzol(Invitrogen,Karlsruhe,Germany).First-strand cDNA was synthesized from100ng total RNA using SuperScript III kit (Invitrogen)according to the manufacturer’s recommendation.For FOXP3 and glyceraldehyde phosphate dehydrogena(GAPDH)transcripts,real-time polymera chain reaction(PCR)was performed with a LightCycler (Roche Diagnostics)bad on specific primers and generalfluorescence detection with SYBR Green.The following primer combinations
were Figure1.Frequency of CD4؉CD25hi T cells.(A)Flow cytometric analysis of CD4 and CD25on peripheral blood-derived T cells of a healthy individual and a patient with CLL.Cells were either stained with the appropriate isotype controls(left panel)or CD4and CD25mAbs(right panel).CD4ϩCD
25ϩT cells were divided into CD25lo and CD25hi cells according to previously published data.25Numbers reprent percent-age of events within the respective rectangle.Settings shown here were ud for the analysis of all samples under study.(B)Frequency of CD4ϩCD25hi T cells in26 control and all73CLL samples.Shown here are median,75percentile(box),SD (whiskers),and outliers(dots)(*PϽ.001,Student t test).(C)Frequency of CD8ϩCD25hi T cells.
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ud:FOXP3forward,5Ј-CGG ACA CTC AAT GAG ATC TA-3Ј;FOXP3 rever,5Ј-ATC CTC CTT TCC TTG ATC TT-3Ј;GAPDH forward, 5Ј-TGA TGA CAT CAA GAA GGT GGT GAA-3Ј;and GAPDH rever, 5Ј-TCC TTG GAG GCC ATG TGG GCC AT-3Ј.All PCRs were performed using LightCycler-FastStart DNA Master SYBR Green I kit(Roche Diagnostics).cDNA from Jurkat cells was ud as a standard and normalization to GAPDH was performed for each sample.Relative fold changes of FOXP3expression in CD4ϩCD25hi T cells were normalized to GAPDH as described.38
Generation of dendritic cells
For T-cell stimulation we generated allogeneic dendritic cells(DCs)as described previously.63Briefly,PBMCs were plated in Iscove modified Dulbecco medium(IMDM)with5mM glutamine and25mM HEPES (N-2-hydroxyethylpiperazine-NЈ-2-ethanesulfonic acid;all from PAA Lab-oratories,Coelbe,Germany)and1%autologous plasma for2hours at37°C. Subquently,the adherent cell fraction was cultured for20hours in RPMI 1640(PAA Laboratories)supplemented with2mM glutamine and1% autologous plasma(DC medium).On day1,new DC medium containing 800U/mL granulocyte-macrophage colony-stimulating factor(GM-CSF; Leucomax;Novartis,Nuremberg,Germany)and1000U/mL IL-4(Immu-notools,Friesoythe,Germany)was added.Cytokines were added on day3 in fresh DC medium.On day5,nonadherent cells were replated in fresh DC medium with cytokines.On day6,10ng/mL tumor necrosis factor␣(TNF-␣),1g/mL prostaglandin E2(PGE2;both from Sigma Aldrich, Taufkirchen,Germany),1000U/mL IL-6,and10ng/mL IL-1(both from R&D Systems)were added and mature DCs were harvested on day7. Asssment of inhibitory function of CD4؉CD25hi T cells
To asss the suppressive activity of CD4ϩCD25hi T cells on conventional T-cell proliferation,a modified allogeneic MLR was performed as previ-ously described.14,64,65Briefly,after magnetic paration both CD4ϩCD25Ϫand CD4ϩCD25hi T cells were incubated for20hours with10U/mL IL-2
and0.5g/mL anti-CD3mAb in X-VIVO15.Subquently,the preactivated CD4ϩCD25ϪT cells(5ϫ104/well)were cocultured with irradiated allogeneic PBMCs(2ϫ105/well)or mature DCs(DCs/T cells, 1:20)in X-VIVO15supplemented with10%fetal calf rum(FCS),100 U/mL penicillin/streptomycin,and2mM glutamine(all from Gibco Invitrogen).Purified allogeneic CD4ϩCD25hi T cells were added at different concentrations as indicated.There was no influence on the inhibitory effect of CD4ϩCD25hi T cells when the CD4ϩCD25ϪT cells (effector T cells)were not preactivated prior to the inhibition assay.On day 4the cells were puld with BrdU and BrdU incorporation was analyzed20 hours later as described(e“CD4ϩT-cell isolation and culture”). Asssment of viability after in vitro incubation with
fludarabine
To measure the effect offludarabine,PBMCs from healthy controls were cultured in RPMI1640supplemented with10%FCS,100U/mL penicillin/ streptomycin,and2mM glutamine with or without10Mfludarabine (Fludara;MedacSchering Onkologie,Munich,Germany)for48hours. Cells were then harvested and stained using the following antibodies: FITC-conjugated CD3,APC-Cy-7–conjugated CD4,PE-Cy-7–conjugated CD25,7-amino-actinomycin D(7-AAD),and PE-conjugated annexin V(all from BD Biosciences)according to the manufacturer’s instructions.Viable cells were de
发动机曲轴
fined as annexin V and7-AAD double-negative cells and listed as percent of parental CD4ϩCD25Ϫor CD4ϩCD25ϩT cells.
Cytometric bead array for chemokines
The concentration of interferon␥(IFN-␥)in cell culture supernatants was measured using the human Th1/Th2cytokine kit II(BD PharMingen).In brief,capture beads were mixed with culture supernatants and PE detection reagent and incubated for3hours at room temperature.The beads were then washed with wash buffer and analyzed.Statistical analysis
different的用法Comparison between paired or unpaired groups was performed using the appropriate Student t test.P values below.05were defined as statistically significant.Due to the explorative nature of this study,no multiplicity adjustment procedures were performed.All statistical analys were performed using the SPSS statistical software package(SPSS12.0for Windows,SPSS,Chicago,IL).
Online supplemental material
Figure S1depicts proliferation of purified CD4ϩT cells from healthy individuals and CLL patients activated with anti-CD3and anti-CD28 mAbs.Table S1contains information on the patients.
Results
Incread frequencies of CD4؉CD25hi T cells in patients
with CLL
Frequencies of CD4ϩCD25hi T reg cells were assd using multi-colorflow cytometry and following previously published data.25As depicted in Figure1A,healthy donors showed a significant number of CD4ϩCD25lo T cells with a smaller percentage of CD4ϩCD25hi T cells,whereas we found an incread frequency of CD4ϩCD25hi T cells in patients with CLL.Using the ttings,we analyzed samples from73patients with CLL and26healthy individuals.The frequency of T reg cells in controls(4.5%Ϯ1.1%)was similar to previously published results(Figure1B).25In contrast,patients with CLL showed significantly incread frequencies of T reg cells (10.4%Ϯ4.4%,PϽ.001).Control experiments asssing cell surface expression of CD25on CD8ϩT cells did not reveal an overall activation of T cells in patients with CLL,supporting an increa in CD4ϩCD25hi T reg cells(Figure1C).
CD4؉CD25hi T cells from patients with CLL also express T reg cell-associated proteins
白萝卜英文
Before evaluating T reg cell function,we assd expression of proteins that have been previously associated with T reg cells.The included CTLA4,GITR,CD62L,TGF-1,IL-10,and FOXP3.The frequency of CD4ϩCD25hi CTLA4ϩT cells was significantly incread in patients with CLL compared to healthy individuals both for intracellular and extracellular staining(Figure2A-B; PϽ.01and PϽ.05for panels A and B,respectively).Similarly, CD4ϩCD25hi GITRϩcells from patients with CLL were signifi-cantly augmented(3.34%Ϯ1.67%in patients versus 0.96%Ϯ0.29%in controls,PϽ.001;Figure2C).GITR expres-sion in conventional CD4ϩCD25ϪT cells was similarly low in both populations(data not shown).In patients with CLL the CD4ϩCD25hi T-cell population also contained incread frequencies of CD62Lϩcells(PϽ.01;Figure2D).
Intracellular expression of TGF-1and IL-10was assd becau the2cytokines have been associated with T reg cell function.66,67Similarly to CTLA4,GITR,and CD62L,TGF-1and IL-10were significantly incread in CD4ϩCD25hi T cells from patients with CLL(Figure2E-F;PϽ.001and PϽ.05in panels E and F,respectively).We also obrved IL-10ϩas well as TGF-1ϩcells within conventional CD4ϩCD25ϪT cells in the patients but not in healthy controls(data not shown).Becau FOXP3has been shown to be a crucial molecule for murine CD4ϩCD25ϩT reg cells,
2020BEYER et al BLOOD,15SEPTEMBER2005⅐VOLUME106,NUMBER6
钢铁侠3英文版we also performed real-time PCR for FOXP3in a subt of patients with CLL and healthy individuals.PCRs showed strong expression of FOXP3mRNA in CD3ϩCD4ϩCD25hi T cells from healthy individuals (n ϭ7)and expression of FOXP3was even higher in the CD3ϩCD4ϩCD25hi T cells from patients with CLL (n ϭ6).Overall,CD4ϩT cells expressing high levels of CD25,FOXP3,CTLA4,GITR,CD62L,TGF-1,and IL-10are significantly incread in patients with CLL.
Reduced inhibitory function of CD4؉CD25hi T cells from patients with CLL
In 18patients with CLL,sufficient numbers of highly purified CD4ϩCD25hi T cells were isolated to analyze their inhibitory function in comparison to T reg cells from 16healthy controls.Regulatory function of CD4ϩCD25hi T cells was assd using an allogeneic MLR of CD4ϩCD25ϪT cells and allogeneic irradiated PBMCs 65or DCs as stimulators.14,64Autologous conventional CD4ϩCD25ϪT cells from patients with CLL were not ud in the MLR becau the T cells might themlves be inhibitory.
In controls,significant inhibition of allogeneic CD4ϩCD25ϪT cells as exemplified in Figure 3A (control,
P Ͻ.01)was deter-mined at 1:1ratios of allogeneic conventional T cells to T reg cells.In contrast,patients with CLL pretreated with fludarabine-containing chemotherapy showed reduced or even abrogated T reg cell function (Figure 3A,flud).T reg cells from patients with CLL never treated with fludarabine-containing therapy regimens showed inhibitory function similar to healthy controls (Figure 3A,w/o flud).Asssment of IFN-␥production by the CD4ϩCD25ϪT cells confirmed the proliferation results.IFN-␥was greatly diminished in cultures inhibited by CD4ϩCD25hi T cells from healthy donors and patients with CLL never treated with fludarabine,but not in cultures derived from patients previously treated with fludarabine (Figure 3B).The data were further corroborated at lower numbers of T reg cells (Figure 3C).Healthy controls and patients with CLL never treated with fludarabine showed inhibition at lower ratios of T reg cells to conventional T cells,whereas patients with CLL pretreated with fludarabine did not show any inhibition at the conditions (Figure 3C).In Figure 3D inhibition of allogeneic T-cell proliferation by T reg cells is shown for all healthy controls and patients at a 1:1ratio of T reg to conventional T cells.Overall,there was a significant reduction of T reg cell-induced inhibition within the fludarabine group (P Ͻ.001),whereas patients with CLL never treated with fludarabine showed inhibitory function of T reg cells that was not significantly different from healthy controls.
Sorting of CD4ϩCD25hi T cells by fluorescence-activated cell sorting (FACS)in some experiments did not reveal different results,further supporting that the cells had lost their inhibitory function (data not shown).U of allogeneic mature DCs instead of PBMCs for T-cell stimulation led to similar results,suggesting that the obrvation was intrinsic to CD4ϩCD25hi T cells derived from patients with CLL (data not shown).Interestingly,when asssing proliferation of CD4ϩT cells upon stimulation with anti-CD3and anti-CD28mAbs,we obrved normal T-cell proliferation in 3of 3patients pretreated with fludarabine,whereas 7of 11patients untreated or not treated with fludarabine showed significantly reduced overall CD4ϩT-cell proliferation (Figure S1).
CD4؉CD25hi T cells are incread in patients with CLL with extended dia
Next we assd the frequency of T reg cells in context of stage of dia in previously untreated patients with CLL (Figure 4).Of all 73patients,26were previously untreated.This analysis revealed a correlation between T reg cell frequency and stage of dia with highest numbers of T reg cells in patients with extended dia (Binet C)followed by patients with Binet B and with lowest but still incread numbers in Binet A.In patients in Binet stage B and especially in Binet stage C we also obrved an increa of CD4ϩCD25hi T cells coexpressing GITR,CD62L,and intracellular CTLA4compared to healthy controls (data not shown).
CD4؉CD25hi T cells are reduced particularly after therapy with fludarabine
Next,we investigated the association of T reg cell frequencies and therapy (Figure 5).For this analysis,we only included samples from patients in Binet stages B and C becau most patients with Binet stage A had not received therapy.Again,untreated patients (Binet B/C)showed an incread frequency of CD4ϩCD25hi T cells (Figure 5A,no tx)coexpressing GITR,CD62L,and intracellular CTLA4(data not shown).Patients treated with chemotherapy excluding fludarabine (w/o flud)showed a similarly incread frequency of T reg cells and there was no correlation of frequency of T reg cells and time from therapy to phenotypic T reg -cell asssment (data not shown).In contrast,asssment of T reg cell counts in patients receiving fludarabine treatment showed significantly re-duced T reg cell frequencies only in patients who had received last fludarabine treatment less than 18months prior to T reg cell asssment (P Ͻ.01).The number of cells coexpressing
GITR,
Figure 2.Expression of proteins associated with T reg cells.CTLA4,GITR,CD62L,TGF-1,and IL-10were assd on CD4ϩT cells coexpressing CD25by either cell surface or intracellular multicolor flow cytometry.Percent positive cells were determined using stringent gating criteria with less than 1%of events within the positive gate when analyzing respective isotype controls.At least 25,000events per analysis were acquired.Each dot reprents a single individual assd in the respective group;mean expression (line)of all samples in each group is also shown.Significant differences (P Ͻ.05,Student t test)between controls and CLL samples are marked by an asterisk:(A)intracellular CTLA4(P Ͻ.01),(B)extracellular CTLA4(P Ͻ.05),(C)intracellular GITR (P Ͻ.001),(D)extracellular CD62L (P Ͻ.01),(E)intracellular TGF-1(P Ͻ.001),and (F)intracellular IL-10(P Ͻ.05).(G)Expression of FOXP3by human CD4ϩCD25hi T cells.CD4ϩCD25hi T cells and CD4ϩCD25ϪT cells were sorted by MACS from peripheral blood of healthy controls (n ϭ7)and patients with CLL (n ϭ6).Real-time PCR for FOXP3was performed and relative fold changes of CD4ϩCD25hi T cells to CD4ϩCD25ϪT cells were normalized to GAPDH as described.38
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CD62L,and intracellular CTLA4was also lower than in patients never treated with fludarabine (data not shown).Tho patients receiving their last fludarabine treatment more than 18months prior to T reg cell measurement showed T reg cell frequencies comparable to patients treated without fludarabine-containing regimens or even untreated patients.It needs to be mentioned that the patient groups treated with fludarabine were enriched for patients with larger tumor burden and progressive dia,whereas many of the untreated patients showed a slower dia progression.In 21patients we obtained blood samples at least at 2different time points more than 6months apart (range,7-128months).At the cond time point we obrved in 5of 6untreated CLL patients slightly incread or similar frequencies of T reg cells (Figure 5B,no
tx).In patients treated with non–fludarabine-bad chemotherapy (w/o flud),there was a diver respon with 2patients showing incread and 4patients lower frequencies.In contrast,for 8of 8patients treated with fludarabine,frequencies were lower after fludarabine therapy (Figure 5B,flud).Overall,frequency of T reg cells in patients with CLL was associated with stage of dia and with significantly reduced frequencies after fludarabine-bad chemotherapy.
To asss whether fludarabine might preferentially induce cell death in CD4ϩCD25ϩT cells we incubated PBMCs with 10
M
Figure 3.Functional analysis of CD4؉CD25hi T cells.Highly purified CD4ϩCD25ϪT cells were stimulated by allogeneic irradiated PBMCs or DCs either in the prence or abnce of highly purified CD4ϩCD25hi T cells derived from patients with CLL or healthy donors (both allogeneic).T reg cells from 16controls,10patients pretreated with fludarabine,and 8patients never treated with fludarabine were assd in the MLRs.As a function of T-cell inhibition,proliferation (A)and IFN-␥production (B)were measured.Panel A shows reprentative experiments from a control,a CLL patient pretreated with fludarabine (flud),and a patient never treated with fludarabine (w/o flud).Open bars (PB)indicate background proliferation of irradiated allogeneic PBMCs;light gray bars (PB ϩT conv ),alloantigen induced proliferation of CD4ϩCD25Ϫconventional T cells;dark gray bars (PB ϩT reg ),background proliferation of CD4ϩCD25hi T reg cells;and black bars (PB ϩT conv ϩT reg ),proliferation of CD4ϩCD25Ϫconventional T cells in the prence of CD4ϩCD25hi T reg cells at a 1:1ratio (error bars reprent SD;*P Ͻ.01Student t test).(B)Measurement of IFN-␥by cytokine bead array in the supernatants from cultures described in panel A.(C)Inhibition of proliferation of CD4ϩCD25Ϫconventional T cells by CD4ϩCD25hi T reg cells at different ratios (responders to suppressors)from a healthy donor (E ),a fludarabine-treated CLL patient (F ),and a CLL patient never
treated with fludarabine (f ).Reprentative experiments are shown here,error bars reprent SD.(D)Percentages of inhibition of proliferation of CD4ϩCD25Ϫconventional T cells by CD4ϩCD25hi T reg cells at a 1:1ratio from all healthy donors (n ϭ16),fludarabine-treated patients (n ϭ10),or patients with CLL never treated with fludarabine (n ϭ8)(*P Ͻ.001Student t
test).
Figure 4.Frequency of CD4؉CD25hi T cells in context of stage of dia in previously untreated CLL patients.Patients were classified according to the Binet classification:Binet A (n ϭ9),Binet B (n ϭ13),and Binet C (n ϭ4).Shown here are median,75percentile (box),SD (whiskers),and outliers (dots)of data obtained by multicolor flow
cytometry.
Figure 5.Correlation of therapy and frequency of CD4؉CD25hi T cells in patients with CLL.(A)Patients treated with fludarabine less than 18months prior to T-cell analysis (flud Ͻ1.5a)were compared with healthy controls (control),untreated (no tx),otherwi treated patients with CLL (w/o flud)or patients with CLL treated with fludarabine more than 18month prior to analysis (flud Ͼ1.5a)for the frequency of CD4ϩCD25hi T cells as assd by multicolor flow cytometry.Shown here are median,75percentile (box),SD (whiskers),and outliers (dots);*P Ͻ.01for flud Ͻ1.5a versus flud Ͼ1.5a by Student t test.(B)Serial analysis of CD4ϩCD25hi T cells for the 3CLL treatment subgroups at 2different time points parated by at least 6months.
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BEYER et al
BLOOD,15SEPTEMBER 2005⅐VOLUME 106,NUMBER 6
