Persistent changes in the immune system 4 10 years after ABMT

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1 Bone Mrrow Trnsplnttion, (1999) 24, Stockton Press All rights reserved /99 $15. Persistent chnges in the immune system 4 yers fter ABMT T Nordøy 1, A Kolstd 1, P Endresen 2, H Holte 3, S Kvløy 3, G Kvlheim 3 nd A Huseekk 4 Deprtments of 1 Oncology, 2 Phrmcology nd 4 Immunology nd Trnsfusion Medicine, University Hospitl of Tromsoe, Tromsoe; nd 3 The Norwegin Rdium Hospitl, Oslo, Norwy Summry: The im of the present study ws to investigte whether the erly chnges in the immune system oserved fter ABMT would persist over yers. Eighty-five ptients with mlignnt lymphom were treted with ABMT in Norwy from 1987 until Of the 46 ptients in CR y 1997, 36 were enrolled in our study. Medin time from ABMT ws 5 yers (4 yers). Immunophenotyping showed n increse in the medin numer of B cells (.35 9 /l in ptients vs.28 9 /l in s), nd decrese in T cells (1.8 vs /l). Furthermore, lower medin count of CD4 T cells (.54 9 /l in ptients vs.87 9 /l in s) resulted in reduced CD4/CD8 rtios (.8 in ptients vs 1.6 in s). The sugroup of CD4 T cells expressing the nive phenotype CD45RA ws 19.5% in ptients vs 38% in s. In contrst, the frction expressing the memory phenotype CD45RO ws higher in the ABMT group (76% vs 54%). When stimulted, lrger frctions of CD3 CD4 cells in ptients produced IFN- (32% vs 16%) or IL-4 (7% vs 1%) compred to s; thus differentition into the functionlly seprte sugroups Th1 nd Th2, with dominnt Th2 response. Our dt further suggest tht the decrese in CD4 T cell counts nd the imlnce etween CD45RA nd CD45RO susets persists 4 yers fter ABMT. Keywords: ABMT; mlignnt lymphom; immune reconstitution nd function within the first months following ABMT. In contrst, the CD4 T lymphocytes hve een found not to recover completely, even s long s 2 yers post trnsplnt in mny dult ptients. 4 The persisting sunorml CD4 T cell counts nd CD4/CD8 rtios my put ptients t risk of developing opportunistic infections nd even recurrence of their mlignncy. The chnges in the immune system over the first few months nd yers fter ABMT nd PBSCT hve een extensively studied. 5,6 It is, however, still n open question whether complete normliztion of CD4 T cells will occur in these ptients. The role of purging one mrrow or peripherl progenitor cells remins controversil issue. It is not cler whether purging is of clinicl enefit to the ptient. Furthermore, purging my influence the recovery of lymphoid cells post trnsplnt. 7 In the present study we investigted immunologicl recovery in n unselected popultion of lymphom ptients, including 36 out of 46 ptients in complete remission who received ABMT in Norwy etween April 1987 nd Decemer Nerly ll the ptients were treted with TBI-contining conditioning regimen supported y purged utologous one mrrow. Our dt indicte tht chnges in the immune system previously reported erly fter ABMT persist even 4 yers post-trnsplnt. Mterils nd methods Myeloltive therpy supported y utologous one mrrow or peripherl lood progenitor cells hs proven n effective tretment for certin hemtologicl mlignncies including relpsed intermedite grde non-hodgkin s lymphom, 1 multiple myelom 2 nd cute myelogenous leukemi. 3 The role of this tretment in solid tumors is still not cler. At present peripherl lood progenitor cells re the most frequent source of stem cells, replcing one mrrow. Hemtologic recovery of neutrophils nd pltelets ppers lmost consistently within 2 weeks fter reinfusion of PBSC. Reconstitution of lymphoid nd immune effector cells occur more slowly, nd my tke months to yers. It hs een consistent finding tht counts of B lymphocytes, NK cells nd CD8 T lymphocytes rech norml levels Correspondence: T Nordøy, Dept. of Oncology, University Hospitl of Tromsoe, 938 Tromsoe, Norwy Received 9 Ferury 1999; ccepted 2 My 1999 Ptients nd s From April 1987 to Decemer 1993 ll 85 cses of mlignnt lymphom requiring ABMT in Norwy were treted t the Norwegin Rdium Hospitl. In Jnury 1997, 46 ptients were live nd in complete remission (CR) nd 39 of these greed to prticipte in this study. Three ptients were withdrwn from the exmintion due to: n intr-crnil insult; nother ws hospitlized with severe cteril infection nd myelodysplsi nd the third hd psychologicl prolems. Thus, 36 ptients were enrolled. The medin ge ws 39.5 yers (18 59 yers) nd medin time since ABMT ws 5 yers (4 yers). Twenty-eight ptients received one mrrow purged y monoclonl ntiodies nd mgnetic eds. This method is descried elsewhere. 8,9 Ech ptient ws compred with n ge- nd sex-mtched recruited from lood donors. Ptient chrcteristics re summrized in Tle 1.

2 874 Tle 1 Ptient chrcteristics Persistent chnges in the immune system fter ABMT T Nordøy et l Mle/femle 2/16 Age t dignosis 32 (14 51) yers Age t ABMT 34 (14 54) yers Age t study 39 (18 59) yers Time fter ABMT 5 (4 ) yers Dignosis T lympholstic lymphom, 1st remission B lympholstic lymphom, 1st remission Burkitt s lymphom, 1st remission Low grde folliculr non-hodgkin s lymphom, 2nd nd lter remissions High-grde non-hodgkin lymphom, 2nd or lter remission Hodgkin s lymphom, 2nd or lter remission Preprtive regimen TBI nd cyclophosfmide BEAC (crmustine, etoposid, cytrin nd cyclophosphmide) Bone mrrow purging Negtive selection of T cells Negtive selection of B cells 7 ptients 8 ptients 3 ptients 8 ptients 5 ptients 5 ptients 33 ptients 3 ptients 8 ptients 2 ptients Dt re given s medin nd rnges. Kiel clssifiction of lymphoms. Six out of 36 ptients received rdiotherpy some time during tretment. Three of these received mntle field irrdition covering medistinum/thymus. Intrcellulr cytokine nlysis PBMC from ptients nd s were seprted from whole lood using vcutiner cell preprtion tues (CPT; Becton Dickinson). Cells were wshed twice in PBS with.1% BSA, nd resuspended t 2 6 cells/ml in RPMI- 164 supplemented with 5% AB serum, streptomycin nd penicillin. The cells were stimulted for 4 h with PMA (25 ng/ml) nd ionomycin (5 ng/ml) in the presence of refeldin-a ( g/ml), n inhiitor of protein secretion inducing cytoplsmic ccumultion of cytokines. This technique ws originlly descried y Jung nd co-workers, modified y Picker et l. 11 Cultured cells were wshed twice in PBS with BSA nd stined with MoAs to the following cell-surfce mrkers: CD3, CD4 nd CD45RO. The cells were then fixed nd permeilized with FACS permeilizing solution ccording to the mnufcturer s instruction (FstImmune Kit; Becton Dickinson), followed y stining with MoAs specific for IL-4 or IFN-. The smples were nlyzed on FACScn flow cytometer using Lysis II or CellQuest softwre (Becton Dickinson) on HP 34 or Mcintosh Qudr worksttion. A tight light sctter gte ws drwn round the mjor lymphocyte popultion. Cells in this gte, stining doule positive for CD4 nd CD3, or doule positive for CD4 nd CD45RO were studied. Ten thousnd events were cquired nd nlyzed from ech smple. Regents/Antiodies Phorol-12-myristte 13-cette (PMA), ionomycin nd refeldin-a (BFA) were purchsed from Sigm (St Louis, MO, USA). MoAs which were used in flow cytometry were FITC-conjugted nti-cd3, -CD4, -CD16, -CD45, -CD45RO, -kpp, nd -lmd; PE-conjugted nti-cd8, -CD14, -CD19, -CD2, -CD45RO, -CD45RA, -CD69, -IL-4, nd -IFN- ; PerCP-conjugted nti-cd3 nd -CD4. All ntiodies were otined from Becton Dickinson (Sn Jose, CA, USA)/PhrMingen (Sn Diego, CA, USA). Immunophenotyping A complete lood count including differentils, ws performed within 6 h fter drwing of EDTA nticogulted lood. Within 24 h, whole lood ws used for immunophenotyping. For most smples, ntiodies were dded to whole lood. After incution, the erythrocytes were lysed nd the smples run in the flow cytometer without further wshing. When testing for immunogloulin light chins on B lymphocytes, the erythrocytes were lysed nd the smple wshed efore stining. Two-color dt were cquired on FACSscn flow cytometer equipped with 488 nm rgon lser (Becton Dickinson). A tight light sctter gte ws drwn round the lymphocytes nd these cells were nlyzed using CellQuest softwre on Power Mcintosh computer. Counts of B cells (CD2 ), T cells (CD3 ), CD4 nd CD8 cells were clculted, using the totl lymphocyte count nd the percentge of cells with the different phenotypes. The CD4CD8 doule-positive group generlly constituted smll popultion ( 3%) of the cells nd ws not counted. The CD4/CD8 rtio ws clculted. Sttisticl methods For comprison of unpired dt etween two groups the Mnn Whitney U test ws pplied. Kruskl Wllis onewy nlysis of vrince ws used for comprison etween severl groups. Coefficients of correltion were clculted y the Spermn rnk test. Dt re given s medins nd rnges if not otherwise stted. P vlues re two-sided nd considered significnt when.5. Results Medin counts of peripherl lood cell There ws no significnt difference etween ptients nd s in numers of leukocytes, thromocytes or red lood cells. The numers of grnulocytes were similr, ut the numers of monocytes nd lymphocytes were lower in ptients (Tle 2). The monocyte count ws.4 9 /l in ptients vs.48 9 /l in s. Lymphocytes were /l in ptients nd /l in s. The difference oserved regrding lymphocytes ws cused y decresed level of T cells (CD3 ) in ptients (1.8 vs /l), while numers of B cells (CD2 ) were incresed (.35 vs.28 9 /l). The numers of nturl killer (NK) cells were similr in the two groups. 16 T lymphocytes Among CD3 lymphocytes CD4 nd CD8 cells constitute the two mjor susets. The numers of CD8 cells were similr in ptients nd s (.65 9 /l vs

3 Tle 2 Medin counts ( 9 /l) nd rnges for cells in peripherl lood of lymphom ptients 4 yers fter ABMT nd s Phenotype Ptients n = 36 Controls n = 36 P vlue (*31) (*32) Grnulocytes ( ) ( ) Monocytes (.2.7) ( ) NK cells (.13.2) (..61) Lymphocytes (.9 5.7) ( ) B cells (CD2 + ) ( ) (.15.61) T cells (CD3 + ) ( ) ( ) CD4 + cells ( ) ( ) CD8 + cells ( ) ( ) CD4/CD8 rtio (.4 1.7) (.5 2.8) Immunophenotyping successful in 31 ptients nd 32 s /l). In contrst, there ws mrked difference in CD4 counts,.54 9 /l in ptients nd.87 9 /l in s (Figure 1). A tendency towrds higher CD4 counts in ptients trnsplnted 7 yers go (.57 9 /l) compred to ptients trnsplnted 4 6 yers go (.47 9 /l), ws oserved. Due to low CD4 cell numer, the CD4/CD8 rtio ws significntly lower mong ABMT ptients (.8) thn s (1.6) (Tle 2). Expression of CD45 isoforms on CD4 T cells Nive CD4 T cells generlly express the high moleculr weight CD45 isoform, CD45RA, wheres memory CD4 T cells hve the low moleculr weight isoform CD45RO. The trnsplnted ptients hd significntly lower frction of CD4 CD45RA cells compred with s, 19.5% vs 38% (Figure 1). The frction of CD4 CD45RO cells ws higher in ptients (76%) thn s (54%) (Figure 1c). In the group we found correltion etween incresing ge nd decresing frction of CD45RA cells, wheres in ptients there ws no such correltion (Figure 2). Immune reconstitution nd one mrrow purging Twenty-eight of 36 ptients received one mrrow purged y negtive selection using MoAs to B cell ntigens (2 ptients) or T cell ntigens (eight ptients) nd immunomgnetic eds. There were no significnt differences in counts of CD4 cells, or CD8 cells oserved etween ptients who received T cell-depleted (eight ptients), B cell-depleted (17 ptients), or unpurged one mrrow (six ptients) (Tle 3). Immunophenotyping ws successful in three out of five ptients with Hodgkin s lymphom. Numers of T cells were not lower mong these ptients. Due to smll numers Persistent chnges in the immune system fter ABMT T Nordøy et l No. of ptients/s No. of ptients/s No. of ptients/s c <.3 >.3.6 >.6 1. >1. No. of CD4+ cells 9/l >5 % of CD4+ CD45RA+ lymphocytes % of CD4+ lymphocytes Figure 1 Distriution of CD4 + cells mong ptients nd s: () totl numers of CD4 + cells; () CD4 + CD45RA + cells; nd (c) CD4 + CD45RO + cells. ( ) Ptients; ( ) s. of ptients with different sutypes of lymphom, results compring different groups re not presented. Production of intrcellulr cytokines Cells from oth ptients nd s were successfully stimulted y PMA nd ionomycin detected y CD69 expression in more thn 9% of the CD4 CD3 cells. There ws significnt difference etween ptients nd s in the percentge of CD4 CD3 cells producing IFN- or IL-4 fter stimultion. In the ABMT group 31.9% ( %) of CD3 CD4 cells produced IFN-, compred to 15.9% ( %) in the group (Figure 3). 875

4 Persistent chnges in the immune system fter ABMT T Nordøy et l 876 % of CD4+CD3+ cells rs =.2 P = NS % of CD4+ cells expressing IFN-γ Ptients totl Ptients totl. % of CD4+CD3+ cells Age rs =.39 P = Age Figure 2 Correltion etween ge nd percentge of CD4 + CD45RA + cells in () ptients nd () s. r s = Spermn correltion coefficient. Tle 3 Medin cell counts ( 9 /l) nd rnges in ABMT ptients who received T or B cell purged or unpurged mrrow (n = 31 ) Cell type B cell purged T cell purged No purge P vlue n = 17 n = 8 n = 6 B cells (CD2 + ) ( ) ( ) (.31.78) T cells (CD3 + ) ( ) ( ) ( ) CD4 + cells ( ) ( ) (.28.61) CD8 + cells ( ) ( ) (.34.77) CD4/CD rtio ( ) ( ) (.5.82) n = 31, immunophenotyping successful in 31 ptients. %CD4+ cells expressing IL ptients totl ptients totl Figure 3 Percentge of CD3 + CD4 + nd CD4 + CD45RO + cells in ptients nd s producing () IFN- nd () IL-4 fter stimultion with PMA nd ionomycin in the presence of refeldin-a. A similr nd even lrger difference ws oserved regrding production of IL-4 y CD3 CD4 cells, 6.9% ( %) vs 1.1% ( %). The ptients hd higher frction of cells of the CD4 CD45RO phenotype. To investigte whether over-representtion of these cells could explin the difference in cytokine profile, we selectively looked t production of cytokines in the CD4 CD45RO suset. The differences ecme less pronounced ut were still significnt. Thirty-five percent ( %) in ptients vs 25% ( %) in s produced IFN-, nd 6.8% ( %) vs 1.7% ( %) produced IL- 4 (Figure 3). Discussion It hs een consistent finding tht the numers of NK cells, B lymphocytes nd CD8 T lymphocytes return to norml levels within the first months following ABMT. In contrst, CD4 T lymphocytes remin sunorml for longer time period. This generlly results in decresed CD4/CD8 rtio for t lest 2 yers post trnsplnt. 12,13 Similr results hve een reported in ptients undergoing intensive chemotherpy without one mrrow or stem cell support. 14,15 Pedrzzini nd collegues 4 reported tht only 14 out of 79 ABMT ptients hd recovered to 4% of norml CD4 T cell counts t medin follow-up of 24 months. They estimted the time to recovery of norml levels of

5 CD4 cells to e 3 4 yers or more. Our dt with medin follow-up of 5 yers show tht the CD4 counts still remin low, t pproximtely 2/3 of levels. We oserved tendency towrds higher CD4 counts mong those who were trnsplnted 7 yers erlier compred to the ptients trnsplnted 4 6 yers erlier. This difference ws not significnt, ut might indicte tht the process towrds normliztion of this popultion of cells continues even fter mny yers. CD4 T lymphocytes generlly express either CD45 high moleculr weight isoform, CD45RA, or low moleculr weight isoform CD45RO. The CD4 CD45RA cells re regrded s nive T cells recently issued from the thymus, wheres the CD4 CD45RO popultion responds to recll ntigens nd represents memory cells. 17 However, new model for immunologicl memory hs recently een presented, postulting tht memory T cells cn revert from the CD4 CD45RO to the CD4 CD45RA nive phenotype in the sence of ntigen. 18 Previous reports hve shown tht the CD4 CD45RA suset is mrkedly reduced during the first months following ABMT nd recovers slowly. 5,19 Hkim nd co-workers 15 clerly demonstrted tht these cells re more sensitive to chemotherpy; their numers dropped severely during nd fter tretment in rest cncer ptients receiving the FLAC (5- FU, leucovorin, doxoruicin, cyclophosphmide) regimen. After 1 yer the numer of CD4 CD45RA cells remined less thn one third of pretretment levels. The CD4 CD45RO suset showed only modest decrese nd recovery ws fster. Our results show tht even 4 yers fter ABMT, the percentge of CD4 CD45RA cells ccounted for 19.5% of CD4 cells in ptients compred to 38% in s. On the other hnd, the CD4 CD45RO suset ws incresed in the ABMT group. Mturtion of T cells hs een shown to decrese s erly s in young dulthood due to involution of the thymus. It hs een postulted tht this prohiits the regenertion of nive CD4 CD45RA cells fter ABMT. 2 In the non-trnsplnt setting Mckll nd collegues 21 oserved tht in dult ptients, depletion of CD4 T cells ws prolonged, compred to peditric ptients. This suggested n inverse correltion etween ge nd the ility to regenerte nive T cells. Mckll nd collegues 22 hve recently pulished dt from mouse models showing tht involuted thymuses from ged nimls could regenerte significnt numer of T cells. They rgue tht ccumultion of memory type CD4 cells fter one mrrow trnsplnttion is not only cused y reduced thymic function in dults, ut lso incresed ntigen-driven peripherl expnsion of nive CD4 CD45RA thymic emigrnts into the CD4 CD45RO suset. Our dt confirm tht the imlnce etween the susets of CD4 cells persists for mny yers fter ABMT, nd it is still n open question whether norml numers of the nive nd memory CD4 T cells cn ever e restored. CD4 T cells produce different cytokines, nd sed on the production profiles two functionlly different sugroups, Th1 nd Th2, cn e defined. Th1 cells minly produce IFN-, IL-2 nd TNF-, wheres Th2 cells produce IL-4, IL-5 nd IL-. 23 Guillume et l 24 nd others 25 hve reported defect in the production of severl cytokines, prticulrly IL-2, erly fter ABMT. In our study higher Persistent chnges in the immune system fter ABMT T Nordøy et l proportion of CD4 T cells produced IL-4 or IFN- fter stimultion with PMA nd ionomycin in ABMT ptients thn in s. Previous studies hve indicted tht IFN- nd IL-4 re minly produced y the CD45RO frction of T cells. 11,26 When djusting for the higher frction of CD4 CD45RO cells in our ptients, the difference ecme less, ut ws still significnt. The reduced numer of CD4 CD45RA cells does not necessrily indicte immune competence, the diversity of the T cell repertoire might e eqully or more importnt. Jerne 27 postulted tht % of the totl repertoire is sufficient for immune competence. Thus, our ptients might e immune reconstituted despite low numer of CD4 T cells. The most physiologicl stimultion of T cells ppers to occur vi the T cell receptor (TcR) in the presence of n ntigen presenting cell. PMA nd ionomycin stimulte ll T cells regrdless of their TcR specificity. Functionl studies of lymphocytes severl yers post-abmt re scrce. As fr s we know, our study is the first to investigte intrcellulr cytokine production t this time point. The dt indicte tht higher frction of CD4 T cells post ABMT re ctivted nd/or hve differentited to ecome Th1 or Th2 cells. The three to four times increse in IL-4 producing cells compred to 5% increse in IFN- producing cells in ptients indictes dominnt Th2 response, t lest fter stimultion with PMA nd ionomycin. Th2 cells re responsile for induction of humorl immunity. We found tht serum levels of IgG, IgA nd IgM were norml in ptients (dt not shown). However, the numers of B cells were incresed. The increse in frctions of Th1 nd Th2 cells in ptients post-abmt could explin why serious opportunistic infections were rre (ptient self reported questionnire, dt not shown), even though the counts of CD4 cells were significntly less tht oserved in s. The ptient mteril in this study is of prticulr interest, ecuse the one mrrows were purged y negtive selection in most cses, using monoclonl ntiodies nd immunomgnetic eds. Furthermore, ll except three ptients received conditioning regimen contining TBI (13 Gy) nd cyclophosphmide (12 mg/kg). This regimen is proly more effective in destroying residul T cells nd B cells in the host thn re other regimens sed on comintion chemotherpy. Anderson nd co-workers 28 reported tht ptients who underwent T cell-depleted ABMT hd profound immunodeficiency not reflected in their phenotypic reconstitution. Pedrzzini et l 4 reported tht purging utologous mrrow with monoclonl ntiodies to B cell ntigens delyed B cell recovery. Keever et l 7 found only modest quntittive nd temporl differences in immune reconstitution in recipients of T celldepleted mrrow compred to recipients of conventionl mrrow grfts. We could not find ny significnt differences etween ptients who received B or T cell-depleted or unpurged one mrrow in medin counts of B cells, CD4 cells, CD8 cells. Ptient numers re low, ut the dt indicte tht lymphocytes present in the grft my ply minor role in the long-term immune reconstitution of these ptients. In conclusion, lymphom ptients in complete remission 4 yers fter ABMT hve mrkedly reduced numer of CD4 T cells, nd especilly the CD4 CD45RA 877

6 878 Persistent chnges in the immune system fter ABMT T Nordøy et l nive T cell comprtment. In ddition, lrger frction of the CD4 T cells produce IFN- or IL-4 upon stimultion with PMA nd ionomycin, suggesting functionl differences compred to norml s. Acknowledgements The technicl ssistnce of Gorn Kuric nd Ann Nyheim is highly pprecited. The ptients re thnked for prticiption. We re grteful for help nd support y the stff t the Stem Cell Lortory t the Norwegin Rdium Hospitl nd in the Bloodnk t the University Hospitl of Tromsoe. The work ws supported y grnts from The Norwegin Cncer Society nd Ern nd Olv Akre. References 1 Philips T, Guglielmi C, Hgeneek A et l. Autologous one mrrow trnsplnttion s compred with slvge chemotherpy in relpses of chemotherpy sensitive non Hodgkin s lymphom. New Engl J Med 1995; 333: Attl M, Hrrousseu JL, Stopp AM et l. A prospective rndomized tril of utologous one mrrow trnsplnttion nd chemotherpy in multiple myelom. New Engl J Med 1996; 335: Zitton RA, Mndelli F, Willemze R et l. Autologous or llogeneic one mrrow trnsplnttion compred with intensive chemotherpy in cute myelogenous leukemi. New Engl J Med 1995; 332: Pedrzzini A, Freedmn AS, Andersen J et l. Anti-B-cell monoclonl ntiody-purged utologous one mrrow trnsplnttion for B-cell non-hodgkin s lymphom; phenotypic reconstitution nd B-cell function. Blood 1989; 74: Storek J, Witherspoon RP, Stor R. T cell reconstitution fter one mrrow trnsplnttion into dult ptients does not resemle T cell development in erly life. Bone Mrrow Trnsplnt 1995; 16: 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 1997; 19: Keever CA, Smll TN, Flomenerg N et l. Immune reconstitution following one mrrow trnsplnttion: comprison of recipients of T-cell depleted mrrow with recipients of conventionl mrrow grfts. Blood 1989; 73: Wng MY, Kvlheim G, Kvloy S et l. An effective immunomgnetic method for one mrrow purging in T cell mlignncies. Bone Mrrow Trnsplnt 1992; 9: Kvlheim G, Holthe H, Jkosen E et l. Immunomgnetic purging of lymphom cells from utogrfts. J Hemtother 1996; 5: Jung T, Schuer U, Heusser C et l. Detection of intrcellulr cytokines y flow cytometry. J Immunol Meth 1993; 159: Picker LJ, Singh MK, Zdrveski Z et l. Direct demonstrtion of cytokine synthesis heterogeneity mong humn memory/ effector T cells y flow cytometry. Blood 1995; 86: Roerts MM, To LB, Gillis D et l. Immune reconstitution following peripherl lood stem cell trnsplnttion, utologous one mrrow trnsplnttion nd llogeneic one mrrow trnplnttion. Bone Mrrow Trnplnt 1993; 12: Scheid C, Pettengell R, Ghielmini M et l. Time-course of the recovery of cellulr immune function fter high-dose chemotherpy nd peripherl lood progenitor cell trnsplnttion for high-grde non-hodgin s lymphom. Bone Mrrow Trnsplnt 1995; 15: Mckll CL, Fleisher TA, Brown MR et l. Lymphocyte depletion during tretment with intensive chemotherpy for cncer. Blood 1994; 84: Hkim FT, Ceped R, Kimei S et l. Constrints on CD4 recovery postchemotherpy in dults: thymic insufficiency nd poptotic decline of expnded peripherl CD4 cells. Blood 1997; 9: Hulstert F, Hnnet I, Deneys V et l. Age-relted chnges in humn lood lymphocyte supopultions. Clin Immunol Immunopthol 1994; 7: Merchensclger M, Terry L, Edwrds R, Beverly PC. Limiting dilution nlysis of prolifertive responses in humn lymphocyte popultions defined y the monoclonl ntiody UCHL 1: implictions for differentil CD45 expression in T cell memory formtion. Eur J Immunol 1988; 18: Bell E, Sprshott SM, Bunce C. CD4 T-cell memory, CD45R susets nd the persistence of ntigen unifying concept. Immunol Tody 1998; 19: Sugit K, Soiffer RJ, Murry C et l. The phenotype nd reconstitution of immunoregultory T cell susets fter T celldepleted llogeneic nd utologous one mrrow trnsplnttion. Trnsplnttion 1994; 27: Hirokw K, Utsuym M, Ksi M et l. Understnding the mechnism of the ge-chnge of thymic function to promote T cell differentition. Immunol Lett 1994; 4: Mckll CL, Fleisher TA, Brown MR et l. Age, thymopoiesis, nd CD4 T-lymphocyte regenertion fter intensive chemotherpy. New Engl J Med 1995; 332: Mckll CL, Punt JA, Morgn P et l. Thymic function in young/old chimers: sustntil thymic T cell regenertive cpcity despite irreversile ge-ssocited thymic involution. Eur J Immunol 1998; 28: Romgnni S. Humn TH1 nd TH2 susets: dout no more. Immunol Tody 1991; 12: 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 1994; 54: Cyeux S, Meuer S, Pezzutto A et l. T-cell ontogeny fter utologous one mrrow trnsplnttion: filure to synthesize interleukin-2 (IL-2) nd lck of CD2- nd CD3-medited prolifertion y oth CD4 nd CD8 cells even in the presence of exogeneous IL-2. Blood 1989; 74: Akr AN, Slmon M, Jnossy G. The synergy etween nive nd memory T cells during ctivtion. Immunol Tody 1991; 12: Jerne N. Idiotype networks nd other preconceived ides. Immunol Rev 1984; 79: Anderson KC, Soiffer R, DeLge R et l. T-cell depleted utologous one mrrow trnsplnttion therpy: nlysis of immune deficiency nd lte complictions. Blood 199; 76: