Immunoregulatory cytokines in bone marrow and peripheral blood stem cell products

Transcription

1 Bone Mrrow Trnsplnttion, (999) 23, Stockton Press All rights reserved /99 $2. Immunoregultory cytokines in one mrrow nd peripherl lood stem cell products RK Singh, K Ino, ML Vrney, DG Heimnn nd JE Tlmdge Deprtment of Pthology nd Microiology, University of Nersk Medicl Center, Omh, NE, USA Summry: In these studies, we compred the phenotype, function, nd expression of type, type 2, nd monocyte-ssocited cytokine mrna trnscripts in utologous one mrrow (BM) nd growth fctor-moilized peripherl lood stem cell (PSC) products. These studies demonstrte tht lymphocytes nd monocytes in stem cell products re normlly ctivted, expressing significntly higher levels of interleukin (IL)-2, 4 nd, interferon gmm (IFN- ), nd tumor necrosis fctor lph (TNF- ), ut not IL-8, s compred to norml peripherl lood mononucler cells (PBMC). In ddition, the levels of, IL- nd TNF- re significntly higher in moilized PSC s compred to BM products. The high cytokine levels re unexpected s T cell function in stem cell products is depressed. PSC products hve high levels of T cell inhiitory ctivity, which directly correltes with IL- expression, oth of which re mechnisms tht might e involved in the immune dysfunction within stem cell products used for utologous stem cell trnsplnttion. These dt demonstrte tht: () immune cells in utologous BM nd PSC products re ctivted with the expression of high levels of type nd type 2 cytokines s well s monokines; (2) PSC products contin high frequency of monocytes which medite T cell inhiitory ctivity; nd (3) despite the high levels of cytokine expression, T cell function in stem cell products is depressed. The significnce of these immune normlities within stem cell products for myeloid nd lymphoid recovery following utologous stem cell trnsplnttion remins to e determined. Keywords: cytokines; stem cells; one mrrow; gene regultion; immunomodultors Moilized PSC products re incresingly used to restore hemtopoiesis fter high-dose chemotherpy (HDT) for solid nd hemtologic mlignncies. 3 This is due in prt to the more rpid myeloid recovery tht occurs following peripherl lood stem cell trnsplnttion (PSCT) with growth fctor-moilized PSC s compred to utologous one mrrow trnsplnttion (BMT). 7 Recent oser- Correspondence: JE Tlmdge, Deprtment of Pthology nd Microiology, University of Nersk Medicl Center, Nersk Medicl Center, Omh NE , USA Received 7 April 998; ccepted 24 July 998 vtions from our lortory nd others hve demonstrted tht there is more rpid immunologicl reconstitution in ptients receiving growth fctor-moilized PSCT s compred to BMT. 7 This might e due to the higher numer of immune effector cells (lymphocytes nd monocytes) infused y PSC s compred to BM products. 8,2 In ddition to the difference in the frequency nd the numer of infused T cells nd monocytes in PSC nd BM products, 2,3 the quntittive difference in the endogenous cytokine production in the different stem cell products nd in the serum post-trnsplnttion hve een suggested to e importnt to lymphohemtopoietic recovery. 4 8 Previous reports hve lso nlyzed the role of immune cells in hemtopoietic recovery nd the expression of growth fctors, such s stem cell fctor, grnulocyte mcrophge colony-stimulting fctor (GM-CSF), nd IL-6. 4 These studies investigted the serum levels of cytokines following trnsplnttion 9 22 with focus on the endogenous levels of hemtopoietic growth fctors s relted to hemtologicl reconstitution. However, the expression of immunoregultory cytokines in stem cell products including PSC nd BM products hs not een exmined. Recent studies hve reported in vitro cytokine production, oth stedy-stte nd induced, y mononucler cells from the peripherl lood (PB) of llogeneic BM trnsplnt ptients post-trnsplnttion nd hve shown defects in the production of, 23,24 IL-6 4 nd IL-. The two primry cellulr components found within stem cell products re lymphocytes nd monocytes. T lymphocytes express two distinct cytokine ptterns. 25,26 Type cells produce nd IFN-, wheres type 2 cells produce, IL-5 nd IL-. The type cytokines promote cellmedited responses while the type 2 cytokines promote immunogloulin production nd inhiit the differentition of type cells nd cytokine relese In ddition, type 2 cytokines cn medite immunosuppression. 3,32 Similrly, monocytes produce IL-, TNF- nd IL-2, which lso modulte the immune response. 33 However, the differentil expression of type, type 2 nd monokine-specific mrna trnscripts nd their role in the regultion of the immune function of mononucler cells from stem cell products remins uncler. In the present study, we nlyzed the phenotype, function, nd stedy-stte expression of cytokine genes in mononucler cells from PSC nd BM products s compred to norml PBMC nd phytohemgglutinin (PHA)- stimulted norml PBMC using semi-quntittive reverse trnscriptse polymerse chin rections (RT-PCR). T cell function ws determined y PHA mitogenic response. In ddition, we determined the presence of cellulr ctivity

2 54 Immune dysfunction in stem cell products tht cn inhiit T cell function. The dt demonstrte tht oth PSC nd BM products express significntly higher levels of type nd type 2 cytokines s compred to norml PBMC. In ddition, growth fctor-moilized PSC products express significntly higher levels of cytokine mrna trnscripts, including, IL- nd TNF- s compred to BM products. However, despite the high levels of cytokine gene expression, T cell function is significntly depressed in oth stem cell products s compred to norml PBMC. Mterils nd methods Ptients Consecutive intermedite-grde non-hodgkin s lymphom (NHL) ptients who were cndidtes for HDT nd PSCT (n = 6, mles nd six femles, medin ge 52, rnge 2 68;) or BMT (n = 2, eight mles nd four femles, medin ge 5, rnge 8 67) t the University of Nersk Medicl Center (UNMC) were entered into this study. Written informed consent for stem cell collection nd utologous trnsplnttion ws otined from ech ptient. All ptients hd received n verge of four cycles of chemotherpy prior to HDT nd stem cell trnsplnttion. PSCT ptients were moilized with grnulocyte colonystimulting fctor (G-CSF) ( g/kg/dy) nd trget dose of mononucler cells/kg ody weight collected nd cryopreserved. BM products were collected from the ilic crest using stndrd procedures. These smples were otined using protocols pproved y the Institutionl Review Bord of UNMC. Mononucler cell isoltion The fresh uncultured BM products from ptients nd PB from norml helthy donors (n = ) were diluted : nd the fresh uncultured PSC products from ptients were diluted :2 in Hnk s lnced slt solution (HBSS) (Gico BRL, Grnd Islnd, NY, USA) nd isolted using Ficoll Hypque (Orgnon Teknik, Durhm, NC, USA). Cells were then wshed twice with HBSS nd djusted to 4 6 cells/ml in RPMI-64 contining % fetl ovine serum (FBS), mm HEPES, gentmycin, nd l-glutmine. Flow cytometric nlysis For phenotypic nlysis, nucleted cells isolted from BM products, PSC products, nd PB were otined following erythrocyte lysis, counted, nd djusted to 6 cells in phosphte-uffered sline (PBS) (Gico BRL) contining 2.5% FBS to mintin cell viility nd processed ccording to the method descried erlier. 8,2,3 Antiodies used in this study were FITC-leled CD3, PE-leled CD4, nd iotin-leled CD8 nd CD4, purchsed from Becton Dickinson (Sn Jose, CA, USA) nd Coulter Corportion (Hileh, FL, USA). All three-color dt were cquired on Becton Dickinson FACStr Plus flow cytometer. Detiled three-color dt nlysis ws performed using the Attrctors nd CellQuest softwre from Becton Dickinson s descried erlier. 8,2,3 Mitogen response The mitogenic response of the mononucler cells isolted from PSC nd BM products s well s PB from norml donors were nlyzed y methods descried erlier. 8 Specific incorportion of 3 H-thymidine in test smples (with PHA) ws compred to control cells (without PHA). Results re presented s stimultion index (SI) clculted using the following formul with counts per minute designted y c.p.m.: Stimultion index = c.p.m. in cultures with PHA (experimentl) c.p.m. in cultures without PHA (control) T cell inhiitory ctivity ssy The methodology to mesure T cell inhiitory ctivity present in the stem cell products nd PBMC from norml donors hs een descried erlier. 8,34 Briefly, 5 responder cells (freshly isolted norml Ficoll Hypque purified peripherl lood lymphocytes (PBL)) were co-cultured with irrdited (5 cgy) puttive inhiitory cells (mononucler cells isolted from BM products, PSC products, or PBMC from norml donors) t different inhiitorto-responder (I:R) rtios in medium contining n optiml concentrtion of PHA (.5 g/ml). Responder cells lone, nd puttive inhiitory cells lone, were lso incuted with medium lone or medium contining PHA (.5 g/ml). Cells were cultured t 37 C nd 5% CO 2 for 96 h. The mitogenic response of the responder popultion ws ssyed y pulsing with Ci/well of 3 H-thymidine over the finl 8 h of culture. Cells were hrvested with 96- well hrvester onto fierglss filters nd the incorportion of 3 H-thymidine ws monitored using Pckrd Multi- Well Bet Counter (Downers Grove, IL, USA). T cell inhiitory ctivity ws determined using the formul: % T cell inhiitory ctivity = c.p.m. in co-cultures of inhiitor plus Responders (I + R) with PHA c.p.m. in responders only with PHA Anlysis of cytokine mrna expression Immeditely following cell isoltion, totl cellulr RNA ws isolted from PSC, BM products, or PBMC using Trizol regent (Gico BRL, Githersurg, MD, USA). 35 Firststrnd cdna ws synthesized y reverse trnscriing 2 g of totl RNA using superscript RT (Gico) ccording to the mnufcturer s protocol. Two microliters of : dilution of first-strnd cdna were dded to 5 l of PCR uffer (Gico BRL), l of dntp mix ( mm ech of dttp, datp, dctp nd dgtp),.5 l of5mm MgCl 2, nd.25 l of Tq DNA polymerse (Gico). Two nd hlf microliters of ech primer ( m ech) were dded to give finl primer concentrtion of.5 m nd the totl volume ws djusted to 5 l with sterile dh 2 O. The mixture ws sujected to DNA mplifiction using DNA therml cycler (Perkin Elmer, Foster City, CA, USA)

3 set t different nneling tempertures (55 C for -ctin,, IL-, TNF- nd IL-8, nd 5 C for nd IFN- ) for totl of 2 cycles for -ctin nd 4 cycles for the other genes. PCR frgments were seprted on 2% grose gel in TAE uffer contining.25 g/ml ethidium romide, visulized nd photogrphed using UV trns-illumintor (Kodk, Rochester, NY, USA). For quntittive nlysis nd to confirm the specificity of the mplified sequences, gels were processed for Southern lot nlysis. Oligonucleotide primers (Tle ) were designed sed on the pulished sequences with 5 primers (+ strnd) nd 3 primers ( strnd) nd flnked y single intron so tht ny mplifiction of contminting genomic DNA would e redily identified. The specificity of the product ws verified y lotting the frgments onto nylon memrnes nd proing with specific oligonucleotide sequences nd nlyzing using digitl utordiogrphy (Phosphor Imger; Moleculr Dynmics, Sunnyvle, CA, USA). Reltive levels of rdioctivity etween smples were determined using Imge Qunt softwre on the Phosphor Imger. Reltive mrna trnscript levels were otined using equl numer of cells with simultneous mplifiction in the liner rnge (see Figure ). Results were not used if the housekeeping gene signls etween groups were undetectle in given smple/experiment. Cytokine signls re expressed s expression index clculted y compring the rtio of ech cytokine signl to the signl from the housekeeping gene -ctin. Tle Cytokine Primers used for RT-PCR nlysis Primers Immune dysfunction in stem cell products Enzyme linked immunosornt ssy (ELISA) The secretory levels of the different cytokines were nlyzed using n ELISA. Primry ntiodies nd iotinylted secondry ntiodies were purchsed from Endogen (Phildelphi, PA, USA) (, IL-, IFN- ) nd R&D Corportion (Minnepolis, MN, USA) (). Cytokines were mesured in culture superntnt following 24 h incution of 5 cells/well with.5 g/ml PHA or without PHA. Culture superntnts were collected nd nlyzed y ELISA. First, l of primry ntiodies (2 g/ml) were coted on Immunolon pltes (Dyntech Lortories, Chntilly, VA, USA), followed y locking uffer (PBS/4% BSA). Fifty microliters of either stndrd (t different dilutions) or test smples nd 5 l of iotinylted nticytokine ntiody were dded to ech well nd incuted for 2 h. Pltes were wshed with wshing uffer (5 mm Tris/.2% Tween) nd l of :8 dilution of vidin-conjugted HRP (Dko Lortories, Crpinteri, CA, USA) dded to ech well. After 3 min, pltes were wshed nd l of TMB sustrte were dded to ech well nd incuted for 3 min. Rections were stopped y ddition of l of.8 m H 2 SO 4 nd the sorency ws determined t 45 nm. Stndrd plots were prepred nd the concentrtion of the unknowns ws determined. Sttisticl nlysis SPSS for Windows (SPSS, Chicgo, IL, USA) ws used for the independent smples t-test (two-tiled) to compre mens, nd the correltion nlysis ws performed using Person s nlysis. The dt presented re composite of ll the ptients nlyzed in ech group. 55 IFN- -ctin TNF- IL- IL-8 IFN- -ctin TNF- IL- 5 -CAA CTC CTG TCT TGC ATT GC-3 5 -GCA TCC TGG TGA GTT TGG G-3 5 -ATG TGC CCG GGA ACT TTG TC-3 5 -GTC TGT TAC GGT CAA CTC G-3 5 -CAG GTC ATT CAG ATG TAG CG-3 5 -GCT TTT CGA AGT CAT CTC G-3 5 -TGA AGT GTG ACG TGG ACA TC-3 5 -ACT CGT CAT ACT CCT GCT TG-3 5 -GAG CTG AGA GAT AAC CAG CTG GTG-3 5 -CAG ATA GAT GGG CTC ATA CCA GGG-3 5 -ATG CCC CAA GCT GAG AAC CAA GAC CCA-3 5 -TCT CAA GGG GCT GGG TCA GCT ATC CCA-3 5 -ACA TAC TCC AAA CCT TTC CAC CC-3 5 -CAA CCC TCT GCA CCC AGT TTT C-3 Proes used with Southern lots 5 -GCA CTT GTC ACA AAC AGT GC-3 5 -GCG ATA TCA CCT TAC AGG AG-3 5 -GCA TCC AAA AGA GTG TGG AG-3 5 -CAA GAT CAT TGC TCC TCC TG-3 5 -CCC TCC ACC CAT GTG CTC CTC-3 5 -CAG GTG AAG AAT GCC TTT AAT AAG CTC CAA CAG AAA GGC ATC TAC AAA GCC ATG AGT GAC TTT GAC ATC-3 Results Immune cells in PSC nd BM products The cellulr phenotypes (T cells: CD3 +, CD4 + or CD8 + cells (low side sctter nd high expression of CD3, CD4 nd CD8), nd monocytes (intermedite side sctter nd high expression of CD4)) in growth fctor-moilized PSC nd BM products from NHL ptients nd PB from norml helthy donors (control) were determined using flow cytometry s descried erlier. 8,2 4 The frequency of CD3 +, CD4 + nd CD8 + cells ws significntly higher in PSC products s compred to BM products (ll P.), ut were similr to norml PB (Figure 2, nd c). In contrst, the frequency of CD3 +, CD4 + nd CD8 + cells ws significntly lower in BM products s compred to norml PB (P =.5) (Figure 2, nd c). The frequency of CD4 + monocytes lso ws significntly higher in PSC products s compred to BM (P.) nd norml PB (P.) (Figure 2d). Immune functions of BM nd PSC products The T cell mitogenic ctivity of the BM nd PSC products ws nlyzed y determining their prolifertive response to PHA nd expressed s SI, s descried erlier. 8,2,34 Norml PBMC were used s seline control in these studies,

4 56 Immune dysfunction in stem cell products IFN-γ IL- IL-8 TNF-α 9 5 IFN-γ : : : : : : : : 6 5 IL : : : : : : : : 7 TNF 8 IL : : : : 6 : : : : Smple dilutions Smple dilutions Figure () A representtive figure demonstrting the linerity of the RT-PCR nlysis for the cytokine mrna expression in PSC product using seril dilution of cdna. Lne, no dilution; lne 2, : dilution; lne 3, : dilution; lne 4, : dilution; lne 5, : dilution. () Quntittive nlysis of the mplifiction t different dilutions. The vlues re generted y ImgeQunt softwre from the Southern lots nd Phosphor imger nlyses.

5 Immune dysfunction in stem cell products % CD3+ cells % CD4+ cells BM PSC PBMC BM PSC PBMC % CD8+ cells c % CD4+ monocytes d BM PSC PBMC BM PSC PBMC Figure 2 The frequency of immune cells in stem cell products (moilized PSC, n = 6; BM, n = 2) nd norml PBMC (n = ) ws nlyzed y immunophenotyping using flow cytometry. The vlues re men stndrd error of the men (s.e.m.) for ech group. () frequency of CD3 + cells; () frequency of CD4 + cells; (c) frequency of CD8 + cells; (d) frequency of CD4 + monocytes. The vlues re men s.e.m. for ech group. Significntly different from BM products (P.5). Significntly different from norml PBMC (P.5). nd the results re shown in Figure 3. In these studies, the PHA mitogenic response of the mononucler cells from PSC products ws significntly higher compred to the mononucler cells from BM products. However, the T cell mitogenic response in oth BM nd PSC products ws significntly lower s compred to norml PBMC, suggesting depressed immune function. Interestingly, the seline levels of prolifertion in the PSC nd BM products were higher s compred to norml PBMC (c.p.m. in untreted cultures: PSC products, ; BM, ; nd norml PBMC, ). Nonetheless, the response Stimultion index PHA response, BM PSC PBMC Figure 3 The mitogenic response in stem cell products (moilized PSC, n = 6; BM, n = 2) nd norml PBMC (n = ). The mitogenic (PHA.5 g/ml) response ws nlyzed nd the stimultion index clculted. The vlues re men s.e.m. for ech group. Significntly different from BM products (P.5). Significntly different from norml PBMC (P.5). to optiml concentrtions of PHA (.5 g/ml) in PSC ( 95 96) nd BM products ( ) ws significntly lower thn tht of norml PBMC ( ), leding to decresed S.I. in BM (.6.3) nd PSC (3.6.7) products compred to norml PBMC ( ). To understnd the mechnism of immune dysfunction, we mesured cellulr T cell inhiitory ctivities in BM nd PSC products y exmining the ility of irrdited cells to inhiit the mitogenic response of llogeneic PBMC to PHA. 8,2 4 A significntly higher level of T cell inhiitory ctivity ws oserved in PSC products (P.) s compred to BM products nd norml PBMC (Tle 2). Similr to our erlier oservtion, 34 the T cell inhiitory ctivity ws dependent upon the I:R rtios (Tle 2). Expression of cytokine genes in BM nd PSC products The reltive undnce of specific mrna trnscripts for type (, IFN- ), nd type 2 (, IL-) cytokines nd monokines (TNF- nd IL-8) were nlyzed y RT- PCR in BM nd PSC products nd compred to norml stedy-stte nd PHA-stimulted PBMC (results re shown in Figures 4 6 s n verge expression index stndrd error of the men (EI s.e.m)). mrna trnscripts in PSC products ( ) were significntly higher (P =.6; 44-fold increse) thn in BM products (.63.8). Furthermore, the levels of mrna trnscripts were significntly higher in oth BM (44-fold) nd PSC products (638-fold) s compred to norml PBMC (.3.24; oth P.). However, the mrna levels in PSC products were similr to PHA ctivted PBMC ( ) (Figure 4). Similrly, IFN- mrna expression in oth BM (.3953

6 58 Tle 2 Smples Immune dysfunction in stem cell products Monocyte-dependent T cell inhiitory ctivity in stem cell products T cell inhiitory ctivity t different I:R rtios 4: 2: :.5: PSC products (n = 6) , , , , BM products (n = 2) Norml PB (n = ) T cell inhiitory ctivity t different I:R rtios. The vlues re men percent of T cell inhiitory ctivity stndrd error of the men (s.e.m.) from ech group. The n identified in the prentheses is the numer of smples studied. Significntly different from BM products (P.5). Significntly different from norml PBMC (P.5). I:R = inhiitor to responders rtio.,. Expression index Expression index....., IFN-γ Expression index Expression index.... IL- Figure 4 Expression of () nd IFN- () cytokine-specific mrna trnscripts in stem cell products (moilized PSC, n = 6; BM, n = 2) nd norml PBMCs (n = ). The mrna expression of nd IFN- ws nlyzed y RT-PCR s descried in Mterils nd methods. The vlues re n expression index clculted y compring the vlues of IL- 2 nd IFN- to the housekeeping gene -ctin. The vlues re men s.e.m. for ech group. Significntly different from BM products (P.5). Significntly different from norml PBMC (P.5). Figure 5 Expression of () nd () IL--specific mrna trnscripts in stem cell products (moilized PSC, n = 6; BM, n = 2) nd norml PBMCs (n = ). The mrna expression for nd IL- ws determined y RT-PCR. The vlues re n expression index clculted y compring the vlues for nd IL- with the housekeeping gene ctin. The vlues re men s.e.m. for ech group. Significntly different from BM products (P.5). Significntly different from norml PBMC (P.5)..283) nd PSC products ( ) ws significntly higher (96.6-fold in BM nd 299-fold higher in PSC products; P =. nd P =.3, respectively) compred to norml PBMC (.49.5) nd ws similr to the PHA-ctivted PBMC ( ) (Figure 4). The expression of IL- nd mrna lso ws studied. PSC products ( ) expressed significntly higher levels of IL--specific mrna trnscripts (9.4-fold; P =.5) s compred to BM products ( ), which were similr to PHA-stimulted PBMC ( ) (Figure 5). In ddition, the IL- mrna levels in oth BM (5.5- fold) nd PSC products (63-fold) were significntly higher (P =.3 nd P =., respectively) s compred to norml PBMC (.337.9). In contrst, the levels of -spe-

7 Immune dysfunction in stem cell products Expression index Expression index. TNF-α,,, IL-8 nd P =., respectively)) (Figure 6). In contrst, there were no significnt differences in the levels of IL-8 gene expression in PSC ( ) nd BM products ( ) (Figure 6). Furthermore, the levels of IL-8-specific mrna trnscripts in PSC nd BM products were significntly lower s compred to the stedy-stte PBMC ( (P =.5 nd P =., respectively)) nd PHA-ctivted PBMC ( (P =. nd P =.3)) (Figure 6). In other studies, we nlyzed the secreted levels of the type nd type 2 cytokines in PSC products nd compred them with norml PBMC nd PHA-ctivted PBMC nd PSC products. Similr to the mrna levels, the secretion of oth type - nd type 2-ssocited cytokines in PSC products ws significntly higher compred to norml PBMC (Figure 7). After ctivtion with PHA, there were significntly higher levels of secreted,, IL-, nd IFN- in the culture superntes of norml PBMC. However, PHA tretment incresed the nd IFN-, ut not IL- 4 nd IL- protein secretion y PSC products (Figure 7). Furthermore, in limited numer of PSC (n = 6) nd norml PBMC (n = 4) smples, treted or untreted with PHA, we nlyzed mrna expression y RT-PCR nd protein secretion y ELISA. In these studies, Person s correltion nlysis reveled direct correltion etween cytokine mrna levels nd protein secretion (r =.43, P.) (Figure 8). 59. Figure 6 Expression of () TNF- nd () IL-8 in stem cell products (moilized PSC, n = 6; BM, n = 2) nd norml PBMC (n = ). The mrna expression for TNF- nd IL-8 ws determined y RT-PCR. The vlues re n expression index clculted y compring the vlues for TNF- nd IL-8 with the housekeeping gene -ctin. The vlues re men s.e.m. for ech group. Significntly different from BM products (P.5). Significntly different from norml PBMC (P.5). Correltion of cytokine genes expression, immune phenotype nd function The PHA mitogenic response directly correlted with the frequency of T cells (CD3 + cells, r =.586, P =.) in stem cell products (Figure 9). In ddition, there ws direct correltion etween T cell inhiitory ctivity nd IL- mrna levels (r =.473; P =.26) (Figure 9). cific mrna trnscripts were not significntly different etween the PSC ( ) nd BM products (.3.3) (Figure 5). However, oth BM (68.4-fold) nd PSC (92-fold) products expressed significntly higher levels (P. nd P., respectively) of -specific mrna trnscripts s compred to norml PBMC (.9.4) nd PHActivted PBMC (.5.8) (Figure 5). These dt suggest tht PSC nd BM products hve mixed type nd type 2 pttern of cytokine gene expression. Furthermore, oth PSC nd BM products express higher levels of ll cytokine mrna trnscripts nlyzed s compred to norml PBMC, suggesting tht these products my contin ctivted lymphocytes (Figures 4 nd 5). We lso determined the expression of two monokines, IL-8 nd TNF-, in PSC nd BM products (Figure 6). The levels of TNF- gene expression in PSC ( ) products were significntly higher (2.6-fold) s compred to BM products ( (P =.5)) nd were similr to PHA-ctivted PBMC ( ). Both BM (3.3-fold) nd PSC (34.8-fold) products expressed significntly higher levels of TNF- mrna trnscripts s compred to norml PBMC ( (P =.5 Discussion In the present study we nlyzed the phenotype, function, nd expression of type, type 2, nd monocyte-ssocited cytokine mrna trnscripts in utologous BM nd growth fctor-moilized PSC products s compred to stedy-stte nd PHA-ctivted norml PBMC. The expression of ll the cytokines nlyzed, except IL-8, ws significntly higher in oth BM nd PSC products s compred to norml PBMC, nd similr to PHA-ctivted PBMC. In contrst, the T cell mitogenic function in oth products ws significntly depressed s compred to norml PBMC. In PSC products, this ws not due to chnge in T cell frequency s it ws similr to norml PBMC. The PSC products demonstrted higher mitogenic response (stimultion index) to PHA s compred to BM products, which might e due to the higher frequency of T cells nd/or higher levels of cytokine gene expression. However, lymphocyte function ws significntly decresed in oth stem cell products s compred to stedy-stte PBMC. Similr to our erlier studies, 34 the present dt demonstrted significntly higher levels of cellulr T cell inhii-

8 Immune dysfunction in stem cell products 6 PSC PSC-PHA PBMC PBMC-PHA,$, PSC PSC-PHA PBMC PBMC-PHA,$ IFN-γ, (pg/ml) PSC PSC-PHA IFN-γ (pg/ml) IL- PSC PSC-PHA PBMC PBMC-PHA,$ PBMC PBMC-PHA,$ (pg/ml) IL- (pg/ml) Figure 7 Levels of secreted cytokines in PSC products (n = 6) nd norml PBMC (n = 6). The levels of,, IL- nd IFN- were determined in the culture superntnts of mononucler cells from PSC products nd norml PBMC using ELISA. The vlues re men s.e.m. otined from triplicte cultures for ech smple. Significntly different from norml PBMC (P.5). Significntly different from PSC products (P.5). $ Significntly different from PHA-treted norml PBMC (P.5). mrna levels (expression index) IFN-γ IL- regression plot Protein secretion (pg/ml) Figure 8 Liner regression nlysis of the expression levels of cytokine mrna s determined y RT-PCR nd protein secretion s nlyzed y ELISA. A significnt correltion (Person s coefficient r =.43; P.) ws oserved etween the expression of mrna nd protein secretion. tory ctivity in PSC products s compred to oth BM products nd stedy-stte norml PBMC. Studies on immunologicl reconstitution fter BMT nd PSCT hve demonstrted mrked depression in immune function. 8,9,2,38,39 These studies suggest significnt role for ccessory cells (minly monocytes) in the recovery of immunologic function. 4 6 Ptients receiving stedy-stte BM products tht hve reduced frequency of ccessory cells hve mrginlly slower immune reconstitution thn those receiving n intct-moilized PSC product, suggesting the role of these cells in immune recovery. 4 6 These dt lso indicte tht so-clled ccessory cells (minly lymphocytes nd monocytes) do not mke mjor contriution to hemtologicl recovery, ut might ply n importnt role in immunologicl reconstitution. 4 6,36,37 Interestingly, none of the studies, to dte, hve exmined the role of cytokine gene expression on immunologic recovery. A defect in cytokine production ws reported following stem cell trnsplnttion for solid tumors nd hemtologicl mlignncies regrdless of BM or PSC origin. Previous reports hve lso nlyzed the role of immune cells in hemtopoietic reconstitution nd the expression of stem cell fctor, GM-CSF nd IL-6. 4,7,8 These studies investigted the serum levels of cytokines following trnsplnttion 9,2 with focus on the endogenous levels of hemtopoietic growth fctors s relted to hemtologicl reconstitution. To dte, ll of the studies exmining in vitro cytokine production, oth stedy-stte nd induced, hve een undertken using the PBMC of llogeneic BM trnsplnt ptients 4 nd hve shown defects in the production of, 23,24 IL-6 4 nd IL-. The present study represents the first detiled study of cytokine gene expression in BM nd growth fctor-moilized PSC products used for utologous stem cell trnsplnttion. To increse the sensitivity of detection nd limit the required smple size, we employed semi-quntittive RT-PCR nlysis for these studies. Confirmtion of these results using the nlysis of cytokines t the protein level demonstrted tht mononucler cells from PSC products secreted higher levels of oth type - nd type 2-ssocited cytokines s compred to mononucler cells from norml PBMC. Furthermore, we oserved n ssocition etween the expression of cytokine mrna levels nd protein secretion. The high levels of nd IL- mrna trnscripts in oth BM nd PSC products my contriute to the immune

9 Immune dysfunction in stem cell products Frequency of CD3+ cells r =.586 P <. ducts contin ctivted T cells whose function is suppressed y the high frequency of ctivted monocytes. 34 In BM products, the immune cells re lso ctivted; however, the lower frequency of T cells s compred to norml PB my e the source of the lower immune function. Acknowledgements This work ws supported in prt y Ntionl Institutes of Helth grnts No. R-CA6593, R29-CA7278 nd Nersk Deprtment of Helth nd Humn Services No IL- mrna levels PHA response r =.473 P = T cell inhiitory ctivity Figure 9 Liner regression nlysis of IL- mrna expression levels nd T cell inhiitory ctivity nd PHA response nd frequency of CD3 + T cells in PSC products (n = 6). Person s correltion coefficient nlysis ws used to determine significnce. dysfunction oserved post-trnsplnt. Furthermore, the expression of high levels of IL- nd TNF- in PSC s compred to BM products might e ssocited with the incresed frequency of monocytes nd the high levels of T cell inhiitory ctivity in the PSC products. These studies suggest correltion etween the expression of IL- nd T cell inhiitory ctivity within stem cell products. A recent report suggested tht IFN- nd TNF- my e directly involved in the inhiition of T cell functions. 4 In this study ntiodies directed ginst TNF- nd IFN- olished the T cell inhiitory ctivity in synergistic mnner. Recent preliminry dt from our lortory suggest the involvement of Fs Fs lignd interction in monocyte-dependent T cell inhiitory ctivity. 42 Additionl studies re in progress to nlyze the expression of cytokine genes in isolted supopultions of immune cells (CD4 +, CD8 + nd CD4 + cells) nd to determine their role in immunologic reconstitution following stem cell trnsplnttion. In ddition, future studies need to exmine the role of cytokine expression in the stem cell product on immune recovery. In summry, significntly different ptterns of cytokine gene expression, cellulr phenotype, nd function occur in the PSC nd BM products. The dt suggest tht PSC pro- References Kessinger A, Armitge JO. The evolving role of utologous peripherl stem cell trnsplnttion following high dose therpy for mlignncies. Blood 99; 77: Kessinger A. Is lood or one mrrow etter? Stem Cells 993; : Kessinger A. Circulting stem cells: wxing hemtopoietic. New Engl J Med 995; 333: Sheridn WP. Trnsplnttion of moilized peripherl lood stem cells: role of filgrstim. J Hemother 994; 3: Brugger W, Heimfeld S, Berenson RJ et l. Reconstitution of hemtopoiesis fter high-dose chemotherpy y utologous progenitor cells generted ex vivo. New Engl J Med 995; 333: Vose JM, Anderson JR, Kessinger A et l. High-dose chemotherpy nd utologous hemtopoietic stem-cell trnsplnttion for ggressive non-hodgkin s lymphom. J Clin Oncol 993; : Henon PR, Ling H, Beck-Wirth G et l. Comprison of hemtopoietic nd immune recovery fter utologous one mrrow or lood stem cell trnsplnts. Bone Mrrow Trnsplnt 992; 9: Tlmdge JE, Reed E, Ino K et l. Rpid immunologic reconstitution following trnsplnttion with moilized peripherl lood stem cells s compred to one mrrow. Bone Mrrow Trnsplnt 997; 9: Roerts MM, To LB, Gillis D et l. Immune reconstitution following peripherl lood stem cell trnsplnttion, utologous one mrrow trnsplnttion nd llogeneic one mrrow trnsplnttion. Bone Mrrow Trnsplnt 993; 2: To LB, Roerts M, Hylock D. Comprison of hemtologicl recovery times nd supportive cre requirements of utologous recovery phse peripherl lood stem cell trnsplnts, utologous one mrrow trnsplnts nd llogeneic one mrrow trnsplnts. Bone Mrrow Trnsplnt 992; 9: Guillume T, Sekhvt M, Ruinstein DB et l. Defective cytokine production following utologous stem cell trnsplnttion for solid tumors nd hemtologic mlignncies regrdless of one mrrow or peripherl origin nd lck of evidence for role for interleukin- in delyed immune reconstitution. Cncer Res 994; 54: Tlmdge JE, Reed EC, Kessinger A et l. Immunologic ttriutes of cytokine moilized peripherl lood stem cells nd recovery following trnsplnttion. Bone Mrrow Trnsplnt 996; 7: 9. 3 Mills KC, Gross TG, Vrney ML et l. Immunologic phenotype nd function in humn one mrrow, lood stem cells nd umilicl cord lood. Bone Mrrow Trnsplnt 996; 8: Kwno Y, Tkue Y, Sito S et l. Grnulocyte colony sti-

10 62 Immune dysfunction in stem cell products multing fctor (G-CSF), mcrophge-csf, interleukin-3, nd interleukin-6 levels in ser from children undergoing lood stem cell utogrfts. Blood 993; 8: Ho AD, Mruym M, Mghzchi A et l. Solule CD4, solule CD8, solule CD25, lymphopoietic recovery, nd endogenous cytokines fter high-dose chemotherpy nd lood stem cell trnsplnttion. Blood 994; 84: Test U, Mrtucci R, Rutell S et l. Autologous stem cell trnsplnttion: relese of erly nd lte cting growth fctors reltes with hemtopoietic ltion nd recovery. Blood 994; 84: Tkmtsu Y, Akshi K, Hrd M et l. Cytokine production y peripherl lood monocytes nd T cells during hemopoietic recovery fter intensive chemotherpy. Br J Hemtol 993; 83: Wtts MJ, Jones HM, Sullivn AM et l. Accessory cells do not contriute to G-CSF or IL-6 production nor to rpid hemtologicl recovery following peripherl lood stem cell trnsplnttion. Br J Hemtol 995; 9: Biocchi GP, Scmi G, Benedetti G et l. Autologous stem cell trnsplnttion: sequentil production of hemtopoietic cytokines underlying grnulocyte recover. Cncer Res 993; 53: Ciro MS, Gilln ER, Weinthl J et l. Decresed endogenous circulting steel fctor (SLF) levels following llogeneic nd utologous BMT: lck of n inverse correltion with post- BMT myeloid engrftment. Bone Mrrow Trnsplnt 993; : Hs R, Gericke G, Witt B et l. Incresed serum levels of grnulocyte colony-stimulting fctor fter utologous one mrrow or lood stem cell trnsplnttion. Exp Hemtol 993; 2: Miksits K, Beyer J, Siegert W. Serum concentrtions of G- CSF during high-dose chemotherpy with utologous stem cell rescue. Bone Mrrow Trnsplnt 993; : Welte K, Cionu N, Moore MAS et l. Defective interleukin- 2 production in ptients fter one mrrow trnsplnttion nd in vitro restortion of defective T lymphocyte prolifertion y highly purified interleukin-2. Blood 984; 64: Schneider LC, Antin JH, Weinstein H et l. Lymphokine profile in one mrrow trnsplnt recipients. Blood 99; 78: Romgnni S. Humn Th nd Th2 susets: regultion of differentition nd role in protection nd immunopthology. Int Arch Allergy Appl Immunol 992; 98: Romgnni S. Humn Th nd Th2 susets dout no more. Immunol Tody 99; 2: Pul WE, Seder RA. Lymphocyte response nd cytokine. Cell 994; 76: Romgnni S. Th nd Th2 T helper cells: functions, regultion, nd role in protection nd disese. Intl J Clin L Res 99; 2: Lederer JA, Liou JS, Todd MD et l. Regultion of cytokine gene expression in T helper cell susets. J Immunol 994; 52: Spgnoli GC, Juretic A, Schultz-Thter E et l. On the reltive roles of nd IL- in genertion of lymphokine ctivted killer cell ctivity. Cell Immunol 993; 46: Ymmur M, Modlin RL, Ohmen JD, Moy R. Locl expression of ntiinflmmtory cytokines in cncer. J Clin Invest 993; 9: Khrkevitz DD, Seito D, Blch GC et l. Chrcteriztion of utologous-specific T helper 2 cells in tumor infiltrting lymphocytes from ptients with metsttic melnom. Int J Cncer 994; 58: Nthn CF. Secretory products of mcrophges. J Clin Invest 987; 79: Ino K, Singh RK, Tlmdge JE. Monocytes from moilized stem cell products induce T-cell poptosis. J Leukocyte Biol 997; 6: Chomczynski P, Scchi N. Single-step method for RNA isoltion y cid gunidinium thiocynte-phenol-chloroform extrction. Anl Biochem 987; 62: Kessinger A, Armitge J, Antmn K (eds). Re-estlishing hemtopoiesis fter dose-intensive therpy with peripherl stem cells. High Dose Cncer Therpy. Willims nd Wilkins: Bltimore, 992, pp Jnssen WE, Smilee RC, Elfenein GJ. A prospective rndomized tril compring lood- nd mrrow-derived stem cells for hemtopoietic replcement following high-dose chemotherpy. J Hemtother 995; 4: Kiesel S, Pezzutto A, Moldenhur G et l. B-cell prolifertive nd differentitive responses fter utologous peripherl lood stem cell or one mrrow trnsplnttion. Blood 988; 72: To LB, Juttner C, Stomski F et l. Immune reconstitution following peripherl lood stem cell utogrfting. Bone Mrrow Trnsplnt 987; 2: 2. 4 Lum LG, Joshi ID, Smith MR et l. Constitutive nd mitogenstimulted cytokine mrna expression y peripherl lood mononucler cells from most utologous nd llogeneic one mrrow trnsplnt recipients is intct. Bone Mrrow Trnsplnt 994; 3: Tomiok H, Sto K, Mw WW, Sito H. The role of tumor necrosis fctor, interferon-gmm, trnsforming growth fctor-et, nd nitric oxide in the expression of immunosuppressive functions of splenic mcrophges induced y Mycocterium vium complex infection. J Leukocyte Biol 995; 58: Singh RK, Ageitos AG, Ino K et l. Fs FsL medited T cell poptosis y monocytes nd peripherl tolernce in ptients undergoing high dose chemotherpy nd stem cell trnsplnttion. Act Hemtol 997; 98 (Suppl. ): 85.