defective cytokine production following autologous stem cell ......gnus stem cell transplantation...

ICANCERRESEARCH54,3800-3807,July 15,1994]

ABSTRACT

A substantial body of evidence accumulated in recent years Indicatesaprotracted delay in immune recOnstitUtiOn following autologous stem cell

tramplanthdon. In order to investigate the cellular basis ofthis phenomenon,pertpheral blood mononuclear cells were studied from recipients of autolo.gnus stem cell transplantation for solid tumors and hematological malignancIes@On stimulation with phytOhemagglUtininand phorbol 12-mydstate 13-acetate, tramplant-dedved peripheral blood mononudear cells demonstratestatistically significant depressed production of interleuldn 3 (IL-3), IL-4,granulocyte-macrophage-colony-stimulating factor, and y4nterferon as compared to normal controls, during the first 6 months following engraftment,which recover to normal levels 6 months or more posttransplant. When theoverall group oftransplant recipients is compared to the control group, thereisastatisticallysignificantlowerproductionofIL-2.Inaddition,nodifferences were observed regardless of the source of the engrafted stem cells,whether from bone marrow alone (autologous bone mamw transplantation),from peripheral blood stem cells alone, or from a combination of autologousbone man'ow transplantation and peripheral blood stem cells. The defect

@stedpast 6 months postengraftment Transplant-derived peripheralblood mononuclear cells were Stimulated with combinations of either phytoheniagglUtinIn plus the calcium ionophore A23187, thereby drcumventingthe requirement for accessory cell function, or with photbol 12-myristate13-acetate plus anti-CD2S monoclonal antibody, mhnlcldng the CD2S.B7 cellsurface-ligand InteractiOn capable of triggering and stabilizing IL-2 genetranscription. In both situations, decreased production of IL-2 as compared

to controls was observed in Individuals within 6 months of transplantatiOn.Quantitative polymerase chain reaction Indicates that decreased transcripdon ofJL-2 mRNA followingtransplantation is not due solelyto a decrease inthe absolute numbers of CIW T-cells but Is secondary to reduced numbersof transcriptcopies per cell. Productionof IL-lOwas found to be decreasedregardless ofwhether the autologous graft was ofbone marrow or peripheralblood origin. These findings are consistent with the conclusion that (a)multiple dysregulations exist in the production of cytokines important inimmune homeostasis; (b)a defect occurs at orpriorto the level of transcripdon of IL-2 mRNA; (c) 11-10 does not play a direct role In the pathogenesisofposttransplantation immunosuppression; and (d) there Is no evidence that

peripheral blood stem cells may be superior to bone mawow-deiived stemcells in accelerating immune reconstitution.

INTRODUCTION

Restoration of normal T-cell function in patients receiving highdose chemotherapy followed by ABMT3 is often delayed for a periodof several months to several years, with, characteristically, diminished

Received 11/1/93; accepted 5/16/94.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the Association Sportive contre Ic Cancer, Fabriques de

Tabac RÃ©unies(Neuchatel, Switzerland), Fondation Maisin, and TÃ©lÃ©viefFNRS.2 To whom requests for reprints should be addressed.

:; The abbreviations used are: ABMT, autologous bone marrow transplantation; IL,

interleukin; PBMC, peripheral blood mononuclear cells; PHA, phytohemagglutinin;PMA, phorbol 12-myristate 13-acetate; PCR, polymerase chain reaction; GM-CSF, gran

absolute T-cell numbers, alterations in distribution of T-cell subsets,defective elaboration of cytokines, and decreased cellular proliferation(1â€”7).Unlike allogeneic bone marrow transplantation, ABMT with orwithout infusion of autologous peripheral blood stem cells involves nohistoincompatibility, no graft-versus-host disease, and no administration of immunosuppressive therapy. The pathogenesis of the defectiveT-cell physiology therefore likely differs in the two types of grafting,at least in part. Although T-cells in autologous transplant recipientshave been observed to have defective production of IL-2, the primaryT-cell growth factor, in response to stimulation with mitogen and tointeraction with both anti-CD3 and anti-CD2 monoclonal antibody(8), it remainsunclearwhatunderliesthe defective signal transductionleading to a blunted rise in intracellular calcium concentration([Ca2@]@)essential for transcription of IL-2 mRNA (9). Defectivecytokine production may arise from decreased transcription and/orinstability of cytokine mRNA itself, or, alternatively from defectivetranslation and reduced cytoplasmic half-life of the polypeptideproduct.

Human IL-lO, a homodimer with a molecular weight of 39,000daltons is produced by CD4@ T-cells, by activated CD8@ cells, byEpstein-Barr virus-transformed lymphoblastoid cell lines, and bymonocytes (10) and has potent inhibitory effects on antigen-specificT-cell proliferation (11, 12). In addition, human IL-lO depresses theproduction of a number of cytokines by peripheral blood mononuclearcells activated by either anti-CD3 or PHA, including IFNy, GM-CSF,IL-3, and IL-2 (13). In vitro, IL-lO inhibits allospecific cytotoxicT-cell responses when allogeneic purified monocytes are used asstimulator cells, while anti-IL-lO antibody enhances the proliferativeresponse, suggesting a role for endogenously produced IL-lO insuppressing proliferation in primary mixed lymphocyte culture (12).Together these observations suggest that IL-lO acts as a strong immunosuppressive factor and that dysregulated overproduction ofIL-b might contribute to the defect in cytokine production and T-cellproliferation seen following autologous transplantation.

Recent evidence has indicated that transplantation of stem cellsderived from peripheral blood may lead to a more accelerated hematological and immunological recovery than that provided by ABMTalone, possibly by providing a higher percentage of differentiatingprogenitor cells for engraftment (14, 15). If that were true, peripheralbood stem cells would be the ideal choice for autologous transpian

tation not only because of the practical ease of harvest as compared tobone marrow-derived cells but also for functional reasons in promoting immune reconstitution.

In order to address these questions and investigate the nature ofimpaired cytokine production and defective T-cell immune responsepostengraftment, we have studied PBMC from recipients of autologous transplantation for solid tumors and hematological malignancies.

BCNU, carmustine; WNy, y-interferon;cDNA, complementary DNA.

3800

Defective Cytokine Production following Autologous Stem Cell Transplantation forSolid Tumors and Hematologic Malignancies Regardless of Bone Marrow orPeripheral Origin and Lack of Evidence for a Role for Interleukin-lO inDelayed Immune Reconstitution'

Thierry Guillaume,2Maryam Sekhavat, Daniel B. Rubinstein, Oussama Hamdan, Pierre Leblanc, andMichel L. SymannLaboratory of Experimental Oncology and Hematology, Catholk University of Louvain, 54 avenue Hippocrate, UCL 54.71, 1200 Brussels, Belgium (T. G., M. S., 0. H.,M. L. S.J, and Division ofHematology/Oncology, Boston University Medical Center, Boston, Massachusetts 02118 [D. B. R., P. L.J

on July 21, 2021. © 1994 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

DEPRESSED CYFOKINE PRODUCFION FOLLOWING ABMT

Following in vitro stimulation, transplantation-derived PBMC areshown to have significantly defective production of a number ofcytokines important in immune homeostasis in the early posttransplantation period. Depressed transcription of IL-2 mRNA is demonstrated in the same early posttransplant group, regardless of the sourceof engrafted stem cells, whether from bone marrow (ABMT) or fromperipheral blood (PBSC), suggesting that there are no qualitativedifferences between the two types of grafts in promoting immunereconstitution. Furthermore, by quantitative PCR, this deficit is shownnot to be related solely to decreased absolute numbers of CD4@ cellsfollowing transplantation but to decreased number of IL-2 transcriptcopies per cell. Transplant-derived PBMC were further stimulatedwith combinations of either PHA plus the calcium ionophore A23187,thereby circumventing the need for accessory cell, or with PMA plusanti-CD28 monoclonal antibody mimicking the CD28-B7 cell surface-ligand interaction capable of triggering and stabilizing IL-2 genetranscription (16). In both of these situations, decreased production ofIL-2 as compared to controls was observed in individuals within 6months of transplantation, suggesting that following engraftment,production of cytokine is dysregulated at more than one step in theearly stages of T-cell activation. Lastly, production of IL-lO wasfound to be decreased regardless of whether the autologous graft wasof bone marrow or peripheral blood origin not only indicating a defectin the mononuclear cells producing IL-lO but also suggesting thatIL-b does not play a direct role in posttransplantation immunosuppression. These in vitro findings have direct in vivo implications infuture stategies of stem cell harvesting and possible therapeutic interventions in an effort to accelerate immune reconstitution followingautologous transplantion.

MATERIALS AND METhODS

Patients and Grafts. A total of66 patients with ages ranging from 14 to 62years (median, 40 years) were grafted for the following malignancies: nonHodgkin's lymphoma (n = 22); small cell carcinoma of the lung (n = 10);breast carcinoma (n 11); Hodgkin's disease (n = 6); acute nonlymphocytic

leukemia (n = 7); acute lymphoblastic leukemia (n = 2); germ cell tumor(n 2); rhabdomyosarcoma (n 1); multiple myeloma (n 1); adeno

carcinoma of unknown origin (n = 2); and undifferentiated carcinoma(n 1).Thirty-six patientsreceivedABMT alone,13receivedboth ABMTand PBSC transplant, while 17 received solely PBSC transplant. Pretransplantation chemotherapy of several regimens consisted primarily of thecombinations cyclophosphamide-BCNU-melphalan, cyclophosphamideBCNU-etoposide, carboplatin-thiotepa-cyclophosphamide, BCNU-etoposide-1-@D-arabinofuranosylcytosine-C-melphalan, or 1-@D-arabinofuranosylcytosine-C-cyclophosphamide plus total body irradiation) Forty-onepatients were studied within 6 months postgrafting (median, 50 daysfollowing transplantation), 25 patients 6 months or more postgrafting(median,654days).A groupof 16age-matchednormalswereselectedascontrols.

Forty to 60 ml of blood were obtained from the transplant subjects or fromthe normal volunteers with the approval of the Ethics Committee of theInstitution. PBMC were isolated from heparinized venous blood by densitygradient on Ficoll-Hypaque (Pharmacia, Piscataway, NJ) and washed twice inRPM! 1640 supplemented with 20% fetal calf serum (Gibco/BRL, Paisley,United Kingdom).

Reagents. PHA (Welcome, Dartford, United Kingdom) was reconstitutedin sterile water for a stock of 1 mg/mI and used for in vitro stimulation at 1

@g/ml.PMA (Sigma Chemical Co., St. Louis, MO) and A23187 (Calbiochem,La Jolla, CA) were dissolved in dimethyl sulfoxide for stock solutions of 10ng4&l and 10 mg/mI, respectively, and used for stimulation at 15 ng/ml and 1

@g/ml,respectively. Anti-CD28 monoclonal antibody (CLB-402) was purchased from Janssens Biochemica (Gee], Belgium).

RNA Extraction and cDNA Synthesis. Total cytoplasmic RNA was isolated from 5 x 106 PBMC by the hot phenol extraction method as described(17). After initial denaturation of the RNA, cDNA was synthesized with

reverse transcriptase (Gibco/BRL, Gaithersburg, MD) and random hexanucleotides (Pharmacia) in a reaction containing 50 mM Tris (jH 8.3), 20 mM KC1,10 mM MgCl2, 5 mM dithiothreitol, and 1 mM concentrations of each deoxynucleotide triphosphate for 1 h at 42Â°C.

PCR and Analysis ofAmplification Products. The product of the reversetranscription reaction was diluted to 100 pA,and 5 @.tlwere used for PCRamplification. Quantitative PCR was carried out by comparing the amplification product using as template the cDNA generated from sample PBMC RNAto that obtained using as template a known cDNA probe derived from thehuman IL-2 gene pTCGF-11 (American Type Culture Collection, Rockville,MD).InordertoclearlydifferentiatethebandsizesofthetwoPCRproducts,a 220-base pairlbal-NheI fragment derived from adenovirus type 2 DNA was

ligated to a XbaI site in exon 3 of the pTCGF-11 IL-2 gene and the modifiedgene inserted into the PstI site of the PGEM-4Z plasmid (Promega, Madison,WI). The plasmid DNA was linearized by digestion with EcoRI, and RNA

synthesis was carried out for 1 h at 37Â°C in 40 mM Tris (pH 7.4), 6 mM

MgCI2, 5 m@dithiothreitol, 4 mMspermidine, 40 units of RNase inhibitor,1 mM concentrations of nucleotide triphosphates, and 11 RNA polymerase(Boehringer-Mannheim, Mannheim, Germany). cDNA was then synthesized as described above. The upstream 20-mer primer (5'-AGTGCACCTAC'VFCAAGTI'C-3') situated in exon 1 and the 20-mer downstreamprimer (5'-GCATATFCACATGAATGT-3') situated in exon 4 yielded aPCR product spanning 2 introns and 2 exons. A range of known concen

trations of the modified pTCGF-1 1 IL-2 template and a constant amount of

each reverse transcribed PBMC cDNA sample were coamplified in thesame tube in a reaction containing 50 mM KC1, 10 mMTris-HC1 (pH 8.3),2.5 mt@tMgCl2, 4 mM deoxynucleotide triphosphates, 15 pmol of eachprimer and Taq DNA polymerase (GibcoIBRL). For quantification of thePCR products, 32P-labeledupstreamprimerwas incorporatedinto each ofthe amplification reactions. 40 pmol of oligonucleotide was end-labeledwith [7-32P]ATP (>5000 Ci/mmol) and T4 polynucleotide kinase (Gibco/BRL) and unincorporated radionucleotide was removed by passage through

a NENSORB column (Dupont, New England Nuclear, Boston, MA). Amplification consisted of 30 cycles of 94Â°Cdenaturation (1.5 mm), 55Â°Cannealing (2 mm), and 72Â°Cextension (3 mm). PCR products of the correctsize were visualized on agarose gels stained with ethidium bromide. Thespecific bands were excised and radioactivity was measured.

Assay for Cytokine Production. PBMC (1 x 106 cells/mI) were slim

ulated with mitogens in 48-h cultures. Supernatants were assayed forproduction of IL-2, IL-3, IL-4, GM-CSF, IFN7, and IL-b in 96-well platesby enzyme-linked immunosorbent assay as recommended by the manufacturer (Amersham International, Amersham, United Kingdom; BiosourceInternational, Camarillo, CA).

Statistical Analysis. Values are expressed as the mean Â± SEM. The

significance of differences between groups was calculated using the Student

Newman-Keuls test.

RESULTS

Production of IL-3, GM-CSF, IL-4, and IFN'y. Results of cytokine production in PBMC derived normals versus that seen in recipients of ABMT or autologous peripheral blood stem cell transplantation is presented in Fig. 1. Not unexpectedly, variability in IL-3production is seen among both the normal controls and the transplantrecipients, depending on what may be called the baseline immunological activation of the individual at the time of stimulation withmitogen. Following stimulation with PHA and PMA, mean values fornormals were 2,067 Â±441 pg/mI for IL-3 (n = 14), 16,997 Â±2,313pg/ml for GM-CSF (n 15), and 8,806 Â±1,417 pg/ml for IFNy(n 15). Mean values for IL-4 was 136 Â±33 (n = 15) followingstimulation with PHA plus PMA and 406 Â±88 pg/ml (n = 15) withPMA plus A23187.

Mean values for PBMC derived from autologous transplant recipients engrafted less than 6 months is shown in Fig. 1. Because theyrepresent only 2 of the group of 13, recipients of both periperal bloodstem cells either alone or in conjunction with autologous bone marroware included together with the larger group that received only autol

3801



DEPRESSED CYTOKINE PRODUCI1ON FOLLOWING ABMT

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Fig. I. Values of IL-3, GM-CSF, IL-4 and IFNy as determined by enzyme-linked immunosorbent assay in the supernatant of mitogen-stimulated PBMC (I X 106cells/ml)at 48h: PHA, 1 @sg/ml;PMA, 15 ng/ml; A23187, 1 @.tg/ml.Cells were derived from normal (C) individuals, patients engrafted less than 6 months (<6M) and patients grafted greater than6 months (>6M). All values are pg/mi.

ogous marrow. Mean values for lectin- and PMA-stimulated lymphocytes showed statistically significant decreases as compared to PBMCderived from normal individuals for IL-3 (676 Â±218 pg/mI; n = 13,P = 0.01 1), for GM-CSF (481 1 Â± 1309 pg/ml; n = 13, P < 0.001),

and for IFNy (3259 Â±1199 pg/ml; n = 10, P = 0.01 1). For IL-4, themean value after stimulation with lectins and PMA was 23.4 Â±11pg/mi (n = 19) which represents a difference statistically significantfrom that of the normal controls (P = 0.001), while that of PMA plusA23187 was 243.7 Â±59.6 pg/ml (n = 19) and was not statisticallydifferent from that of the control group (P = 0.125).

The corresponding values for the group of patients longer than 6months following engraftment are depicted in Fig. 1. As in theprevious analysis, the small number of patients who received ABMTplus PBSC or PBSC only are considered together with those whoreceived autologous marrow only.

Mean values for lectin- and PMA-stimulated lymphocytes were1418 Â±417pg/mlforlL-3(n = l8)and7640 Â±2039pg/mlforIFN'y(n 16), which are not statistically different from the values obtainedfrom the normal controls (P = 0.298 and P = 0.646, respectively).However, the mean value of GM-CSF in the supematants of the PHAplus PMA-stimulated PBMC derived from patients grafted for morethan 6 months was significantly higher than the normal stimulatedPBMC (36,540 Â±6, 186 versus 16,997 Â±2,3 13 pg/mi; P = 0.002).PHA plus PMA stimulation in this group yielded a mean IL-4 valueof 100 Â±27 pg/ml (n = 19) and PMA plus A23187 yielded a meanIL-4 value of 435 Â±87 pg/mi (n = 17), neither statistically differentfrom the normal group (P = 0.399 and P = 0.820).

IL-2 Production in the Presence of Lectin, Phorbol Ester, Calcium lonophore, and Anti-CD2S. Among the groupof normalcontrols, mean IL-2 production following stimulation of PBMC withPHA plus PMA for 48 h was 48,120 Â±8,346 pg/mi (n = 15) (Fig. 2).When the overall group of transplanted patients is compared to the

control group (n = 63), there is a statistically significant lowerproduction of IL-2 (12,004 Â±2,138; P < 0.001) although somepatients had normal values. The type of autologous grafting for thoseengrafted for less than 6 months (ABMT alone, n = 15; PBSC alone,n = 14; or combination of ABMT and PBSC, n = 9) does notsubstantially influence the production of IL-2, since there were nostatistical differences in the mean IL-2 production among the 3subgroups (8771 Â±3124, 3822 Â±1589, 6668 Â±1943; P > 0.05).When patients are compared according to the time postgrafting, independently of the type of grafting, there is no statistical differencebetween those engrafted for less than 6 months (n = 38) or longerthan 6 months (n = 25).

Following stimulation with PMA plus A23187, the mean IL-2 valuewas 123,780 Â± 44,390 pg/mi (n = 15), while the mean valuefollowing stimulation with PMA plus anti-CD28 was 12,813 Â±7,280(n 11) for the normal controls. Mean IL-2 values following stimulation with PMA plus A23l87 in PBMC derived from transplantrecipients less than 6 months postengraftment (ABMT, ABMT plusPBSC, and PBSC) was 20,877 Â±5,762 (n = 14; P 0.035) and withPMA plus anti-CD28 were 2,672 Â±529 (n = 17; P < 0.001). Thecorresponding values in patients engrafted for longer than 6 months

were 48,973 Â±8,978 pg/mi (n = 17,P 0.090) and 8,059 Â±2,534pg/mi (n = 15; P = 0.188), respectively, which were not statisticallydifferent from those of normal controls.

IL-b Production in the Presence of Lectin Plus Phorbol Ester.Production of IL-lO by PBMC was assessed following 48 h ofstimulation with PHA plus PMA (Fig. 3). The mean production bycontrol PBMC was 620 Â±161 pg/mi (n = 13). For the transplantedindividuals engrafted less than 6 months considered as a wholewithout subcategorization, the value was 72 Â±15 (n = 63) which isstatistically significantly lower than the control group (P < 0.00 1).This value was not statistically significantly different from the value

3802



DEPRESSED CY@OKINEPRODUCTION FOLLOWING ABMT

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Fig. 2. IL-2 production by PBMC in the presence of lectin, phorbol ester, calcium ionophore and anti-CD28: PHA 1 @g/ml,PMA l5ng/ml, A23187 1 @tg/ml.Values for PBMCstimulated with PHA +PMA are grouped according to the type of autologous grafting (ABMT alone versus ABMT+PBSC versus PBSC alone). Values for PBMC stimulated withPMA + either calcium ionophore or anti-CD28 are presented according to the time post engraftment: less than 6 months (<6M), longer than 6 months (>6M). All values are pg/mi.

obtained in the group engrafted longer than 6 months (101 Â±32pg/mi; n = 23). It should be noted, however, that several patients hada extremely very low values, while some had values in the range ofnormal contols. Furthermore, among patients engrafted less than 6months, no differences were seen as a function of the source ofengrafted cells. Comparison of IL-lO production between the subgroups of autotransplanted patients grafted for less than 6 months,ABMT alone (50 Â±27 pg/mi; n = 35) versus ABMT plus PBSC(75 Â±43 pg/ml; n = 11) and versus PBSC (48 Â±17 pg/ml; n = 13)showed no statistically significant differences significant difference(P = 0.620; P = 0.958).

Transcription of IL-2 mRNA following Transplantation. Consistent with the observed depressed constitutive production of IL-2 inautograft recipients, Northern blot analysis of unamplified mRNAextracted from 5 X 106 PBMC derived from study subjects yielded nodetectable IL-2 message, although the presence of intact mRNA wasconfirmed by hybridization with an actin gene probe (not shown). Toovercome this, as well as the unavailability of large numbers ofPBMC from transplant recipients, IL-2 mRNA was amplified by PCR.To quantify the amplification product, a concurrent amplification wasrun in the same tube, using as template a known amount of asize-modified IL-2 gene easily differentiable on fractionating gelsfrom the sample amplification product. Both the sample and indicatorproducts incorporated upstream primers labeled with [y-32PJATP. Forquantification, a range of known amounts of indicator amplificationproduct was compared to a constant amount of sample amplificationproduct. Characteristic results are shown in Fig. 4. The expectedmolecular sizes of 546 base pairs for the indicator product and 326base pairs for the sample product are indicated. By relating the amountof extracted mRNA used for generating PCR template cDNA to the

known number of source cells, the number of copies of transcribedIL-2 mRNA per PBMC derived from either normal controls or transplant recipients could be calculated (Table 1). For the 5 normalcontrols analyzed, the number varied between 100 and 75OIPBMC,while for the PBMC derived from autotransplanted recipients thevalues varied from 5 to 100IPBMC. By flow cytometric determinationof CD4@ cells, the expression of IL-2 mRNA per CD4@ cells in thetransplanted patients was calculated to vary between 13 and 714copies/cell assuming that the CD4@ cells are the major producersof IL-2.

DISCUSSION

High-dose chemotherapy and transplantation of autologous bonemarrow transplantation and/or peripheral blood progenitor cells havein recent years been investigated as consolidation following standarddose induction therapy in an effort to improve long-term disease-freesurvival in selected patients with lymphoma (18, 19), with germ celltumors (20), and to increase the complete response rates in patientswith metastatic breast carcinoma (21). However, this approach isfrequently associated with an immunodeficiency affecting primarilycellular immunity. Paradoxically, defects have been reported to bemore profound in autologous transplantation as compared to aliogeneic bone marrow transplantation in in vitro tests for both lymphokine-secreting helper function and IL-2-dependent cytotoxic function

(7).In order to further characterize the nature of this defect, we have

analyzed the in vitro production of a number of immunoregulatorycytokines by PBMC derived from autologous transplant recipients.For the purpose of comparison of overall cytokine production in

3803



DEPRESSED CYTOKINE PRODUCTION FOLLOWING ABMT

tion of IL-4 by PBMC following allogeneic bone marrow transplantation (22, 23) while IL-3 and GM-CSF gene expression by Northernblot analysis has been found to be impaired in this group of subjects(24). Nevertheless, the two situations are not directly comparable.While allogeneic transplantation is complicated by the presence ofgraft versus host reactions and the administration of immunosuppressive agents to prevent it, immune reconstitution following autologousrepresents a truer reflection of unmanipulated immune ontogeny. Inthat context, in the present study we report decreased production ofIL-3, GM-CSF, IL-4, and IFN'y following PHA and PMA stimulationof PBMC derived from recipients engrafted for less than 6 months.Furthermore, when stimulated with PMA and a calcium ionophore,IL-4 production was only partially corrected.

Our results indicating depressed production of IFNy in mitogenstimulated autologous transplant-derived lymphocytes differs somewhat from those reported by Heslop (25, 26) who found a preservedability of both allogeneic and autologous transplant-derived lymphocytes to augment both IFN-y and tumor necrosis factor secretion in

vitro in response to added IL-2 to a degree greater than in lymphocytes derived from individuals treated with chemotherapy alone. Thisapparent discrepancy is explicable by the fact that mitogen and IL-2act at different stages of lymphocyte activation. In fact, in that studythe (CEW/CD8@) CD3@ population was found on addition of IL-2 tobe an important source of IFN-y, and it is precisely the IL-2-producingpopulation, principally CD4@ T-cells, which we find to be defectivein response to mitogen and either calcium ionophore or anti-CD28(Fig. 2). Bypassing this defect by therapeutic administration of IL-2 totransplant recipients may in fact serve to at least partially overcomethe early blocks in T-cell activation which we demonstrate here.

In analysis of IL-2 production, autologous transplant recipientswere analyzed in three groups: those receiving bone marrow alone;those receiving bone marrow plus PBSC; and those receiving PBSCalone. The rationale for doing so arises from the suggestion thattransplantation of PBSC might provide a more accelerated hematological recovery compared to that observed following ABMT (14, 15),and that this could be due to the grafting of more differentiatedprogenitor cells. Once engrafted, PBSC-derived transplants mightthen be expected to achieve immune reconstitution more efficientlythan ABMT-derived lymphocytes as reflected in their ability to produce IL-2, the principal T-cell cytokine. In fact, we found PBMC fromautologous transplant recipients of less than 6 months to be defectivein IL-2 production in response to in vitro stimulation by mitogen andthat, in addition, no significant differences existed between thosereceiving ABMT alone, those receiving PBSC alone, and those re

ceiving both.Likewise, comparison of production of IL-lO by the three groups

shows equally depressed levels (Fig. 3), suggesting that stem cells arequalitatively the same from either source and that, significantly, theremay be no biological advantage of accelerated reconstitution in theuse of peripheral blood progenitor cells.

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Fig. 3. Production of IL- 10 by PBMC following stimulation with PHA (I @.sg/ml)plusPMA (15 ng/ml). Values shown are those of the normal control group (C), the overallgroup ofpatients engrafted less than 6 months (<6M), and the group engrafted greater than6 months (>-OM). All values are pg/mi.

autologous transplantation to previous studies in allogeneic engraftment, the recipients were considered here as a group, regardless ofwhether they received bone marrow alone, bone marrow plus PBSC,or PBSC alone. Furthermore, transplant recipients were divided intoan early group (less than 6 months postengraftment) and a late group(longer than 6 months postengraftment) The rationale for doing soresides in both the protracted and individual heterogeneity in immunereconstitution following transplantation which can require from several months to a full year, as well as a direct means of comparison toa previously analysed group of allogeneic transplant recipients (22).Our findings indicate that during the first 6 months following ABMT,the ability of T-cells to produce cytokines in vitro is markedly impaired. An analogous defect has been reported in the in vitro produc

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U?Fig. 4. PCR amplification using as templatescDNA of the size-modified competitor IL-2 geneand reverse-transcribed RNAS of the sample PBMC.The two template cDNAs were amplified simultaneously for 30 cycles in the same tube with identicalsets of primers, as outlined in â€œMaterialsand Methoth.â€•Right, molecular weight. bp, base pairs.

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3804



Table 1 Quantitative determination ofIL-2 mRI'IAcopies per cell in PBMC derived from control and autotransplated individuals following 6 hours in vitro stimulationwithPHA+PMACalculations

are derived from the incorporation of -y-32P-labeled IL-2 5' PCR primer into 30-cycle amplifications of a range of known quantities of size-altered indicator IL-2genetemplatecompared with incorporation into amplification using as template reverse-transcribed eDNA prepared from RNA extracted from known numbers of sample PBMC(seeâ€œMaterials

and Methodsâ€•).Timepostgrafting

No. of copies' % of No. ofcopies/DiseaseType of grafting (days) PBMC CD4/CD8 CD4cellNormal

controlsNil100Nl2750Nl3300N14600N15

200PatientsDel

BCâ€• PBSC 5010LabNHI. ABMT 101 25 32/â€”78DebSCLC ABM'F 158 5 14/6436LamAML ABMT + PBSC 208 5 34/3714SouALL ABMT 257 25 12/25208JulHD ABMT 28810DeNGT ABMT+ PBSC 29715MaeSCLC ABMT 360 10 27/69370H14NHL PBSC 493 100 14/13714DieM PBSC 510 25 24/-104SriNHL ABMT 715 5 30/â€”13TreNHL ABMT 1056 5 19/4026a

@ breast carcinoma; NIL non-Hodgkin's lymphoma; SCLC, small cell lung carcinoma; AML, acute myelogenous leukemia; ALL, acute lymphoblastic leukemia;HD,Hodgkin's

disease; GT, germ cell tumor; M, multiple myeloma.

DEPRESSEDCY@OKINEPRODUCI1ONFOLLOWINGABMT

Addition of phorbol ester, bypassing the requirement for accessorycell, fails to stimulate IL-2 production to levels comparable to normalcontrols in all three groups of recipients following engraftment. Whencells were stimulated with PMA and anti-CD28, IL-2 production waslikewise found to be decreased in recipients engrafted for less than 6months, suggesting that either engagement of the CD28-B7 ligand isdysfunctional in transplanted T-cells or that even if engaged, CD28-B7-mediated stabilization of IL-2 transcription and production isquantitatively defective. These findings corroborate those recentlyreported indicating not only a quantitative decrease in the numbers ofCD3@ T-cells expressing CD28 in the first 12 months followingautotransplantation but also suppressed CD28 proliferation and IL-2secretion in response to B7 or anti-CD28 (27).These results and thefact that monocytes obtained from healthy individuals cannot restorenormal T-cell function in recipients of ABMT (6) suggest that theaccessory cells are not responsible in themselves for the observedimpaired immune reconstitution. Although stimulation of T-lymphocytes through their specific receptor by agents that mimic T-cellreceptor occupancy in the absence of costimulation may result in anonresponsive state (28), our findings indicate that in the case ofautologous transplantation, the defect is an intrinsic one involved theT-cells themselves and not the absence of the costimulatory signalsince if the latter is provided or bypassed, one cannnot overcome thedefective production of IL-2 or the functional dysregulation of theT-cell itself.

Previous studies have demonstrated that following mitogen stimulation normal T-cells augment their production of IL-2 in a heterogeneous manner, wherein some, but not all cells increase cytokineproduction (29). In an analogous demonstration, we have shown thereduced concentration of intracellular calcium following concanavalinA stimulation in recipients ofABMT to be heterogeneous. While thereis an observable increase in the nonresponding cell population, asubpopulation of T-cells that do respond normally persists (9). Itremains unclear whether this cellular heterogeneity translates to areduced production of IL-2 following autologous transplantation andwhether there is an increased proportion of nonresponding T-cellswith persistence of a subpopulation of functional cells, or, alternatively, the entire population is uniformly affected. Whichever the case

our demonstration by quantitative PCR of depressed transcription ofIL-2 mRNA per PBMC suggests that the decreased production of IL-2following autologous transplantation is not due solely to a fall inabsolute numbers of CD4@ T-cells (Table 1), itself a well-describedphenomenon implicated in the delayed immune reconstitution follow

ing engraftment (6). Single cell in situ hybridization of posttransplantation lymphocytes is in progress in an attempt to further clarify thequestion of functional heterogeneity of engrafted T-cells.4

Amplification by PCR was chosen because the low number ofperipheral blood mononuclear cells available from patients havingundergone ABMT and the low transcript copy number per cell hamperdetection of cytokine mRNA by standard Northern or dot blot hybridization. A number of different approaches have been developed toquantify gene expression (30â€”34). However, because even minuteamounts of template mRNA can be artifactually amplified and detected, the very sensitivity of quantitative PCR has contributed to thedifficulty of the technique. Coamplification of the reverse transcribedIL-2 mRNA derived from transplantation subject PBMC with knownamounts of size-altered but otherwise identical indicator human IL-2gene serving as an internal standard was carried out in an adaptationof the technique described by Giiland (31). Amplifying both templates simultaneously in the same tube with identical primers consistently yielded the two PCR product of expected molecular masses,546 base pairs for the altered indicator gene and 326 base pairs for thePBMC sample-derived reverse-transcribed RNA (Fig. 4). By runningappropriate parallel negative controls, direct and reliable radioactivelylabeled quantitative correlation of the two amplification products wasobtained, from which the number of copies of IL-2 mRNA per PBMCwas derived (Table 1), demonstrating the reduced transcription of IL-2in transplant-derived T-cells as compared to normal controls.

A number of recent studies have investigated serum cytokine levelsfollowing autologous transplantation. Baiocchi et aL (35) described asequential coordinated pattern of production of GM-CSF, G-CSF,IL-la, IL-3, IL-6, and IL-8 in the first 23 days following engraftmentand a parallel serum appearance of G-CSF and IL-8 and neutrophil

4 T. Guillaume et aL, manuscript in preparation.

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DEPRESSED CYTOKINE PRODUCtION FOLLOWING ABMT

mice, bone marrow cells injected into unirradiated syngeneic recipients successfully implant into bone marrow, suggesting that bonemarrow elements, unhindered by cytotoxic therapy, effectively repopulate the hematopoietic system (45). Nevertheless, the numericalreimplantation is not tantamount to functional immunological reconstitution. Further studies in the murine model and/or in humansubjects having received variable rounds of pretransplantation cytotoxic therapy may help to answer these questions and contribute todevising optimal schedules in an attempt to collect maximally functional lymphocytes and accelerate immune reconstitution followingtransplantation.

ACKNOWLEDGMENTS

We wish to thank Drs. A. Bosly, C. Doyen, and Y. Humblet for providingblood samples.

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recovery (35). Similar studies have documented the endogeneousproduction of growth factors following autologous transplantation atlevels commensurate with hematological reconstitution in adults (36â€”38) and in children (39) However, to date, all studies of in vitrocytokine production, both constitutional and stimulated, have beendone in allogeneic transplants, showing defects in production of IL-2(8, 23),of IL-3 andGM-CSF(24),andof IL-6 andtumornecrosisfactor a (40). Immune reconstitution following allogeneic transplantation involves the effects of both histoincompatibility and immunosuppressive drugs administered to combat it. Autologous transplantation, entailing neither of the two, offers a more direct, unmanipulatedpicture of immune ontogeny. The present study represents the firstdetailed study of in vitro cytokine production following autologoustransplantation.

PBMC used in these studies were obtained from transplant recipients following bone marrow recovery. Therefore, assessments ofcytokine production were not designed to be prognostic values inpredicting transplantation outcome but rather a mechanistic indicatorof defective immune reconstitution following successful autologousengraftment. Furthermore, by design, solely in vitro studies werecarried out in order to avoid variations in in vivo serum cytokineproduction that may be induced by factors unrelated to the graft itself,such as infection or administered agents. In order to isolate andcharacterize the posttransplantation T-cell dysfunction in vitro, weused mitogen acting on T-cells and not on other T-cell lineages.

The mechanism of inhibition by IL-lO on antigen-specific T-cellproliferation and on cytokine production is both by a direct negativeeffect on T-cells (12) and indirectly by inhibiting the antigen-presenting capacity of monocytes, but not Epstein-Barr virus-transformedlymphoblastoid cells, through down-regulation of class II MHC cxpression (41). The decreased T-cell proliferative response to antigenpresenting monocytes pre-incubated with IL-lO is accompanied by adecreased ability to mobilize intracellular calcium and is only partiallyrestored by high concentrations of exogeneous IL-2 (41). In light ofthese functional considerations and because of the previously suggested role of increased IL-lO levels in the pathogenesis of humanimmunodeficiency virus-associated immunosuppression (42) IL-lOproduction was analyzed in the defection immune reconstitution oftransplantation recipients.

When production of IL-lO was compared in the three groups ofautologous transplanted recipients engrafted for less than 6 months(ABMT alone versus ABMT plus PBSC versus PBSC alone) nodifferences were found. Furthermore, the defect persists in a statistically significant manner in recipients engrafted for longer than 6months (Fig. 3). This observation suggests that the demonstrateddepression in IL-2 production is not related to suppression by IL-lO.The absence of quantitative difference between the transplant cells,whether from bone marrow or from peripheral blood, suggests one oftwo possibilities: either the immune defect following autologousgrafting of hematopoietic stem cells is independent of the cell source;or, alternatively, changes induced by prior chemotherapy have effacedsuch potential differences. In vitro observations have suggested thatprimitive hematopoietic stem cells and long-term hematopoiesis maybe compromised to varying degrees in recipients of cytotoxic agentscommonly used in either standard multiagent chemotherapy or inspecific bone marrow-conditioning regimens (43), while other cvidence has implicated cyclophosphamide-induced changes in bonemarrow endothelium and alterations in the traffic of transplanted cellsinto the hematopoietic compartment (44). Furthermore, in vivo cvidence exists that myeloablative therapy is at least partially responsiblefor posttransplantation immune defects, because such defects areobserved following infusion of normal bone marrow in allogeneictransplantation, even after immunosuppression is discontinued. In

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1994;54:3800-3807. Cancer Res Thierry Guillaume, Maryam Sekhavat, Daniel B. Rubinstein, et al. Immune Reconstitutionand Lack of Evidence for a Role for Interleukin-10 in Delayed Malignancies Regardless of Bone Marrow or Peripheral OriginTransplantation for Solid Tumors and Hematologic Defective Cytokine Production following Autologous Stem Cell

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