From www.bloodjournal.org by guest on December 22, 2014. For personal use only. A Case of Hypereosinophilic Syndrome Is Associated With the Expansion of a CD3-CD4+ T-cell Population Able to Secrete Large Amounts of Interleukin-5 By Duilio Brugnoni, Paolo Airb, Giuseppe Rossi, Aiessandra Bettinardi, Hans-Uwe Simon, Lucia Garza, Cinzia Tosoni, Roberto Cattaneo, Kurt Blaser, and Alessandra Tucci Interleukin-5 (IL-5) is the major soluble factor able to mediate hypereosinophilia.Wereportacaseofhypereosinophilic syndrome in which the presence of population a of CD3-CD4+ cells able to overproduce 11-5 was shown. The lack of CD3 and TCRAB membrane expression on otherwise phenotypically normal mature T lymphocytes together with the absenceofdetectableTCRBVmRNAandclonalrearrangementofTCRBgenesuggested that the abnormal lymphocyte population was the expression of a peripheral T-cell lymphomawith an indolent clinical course. Peripheral blood lymphocytes enriched in this population were able to secretehighlevelsof11-5 but not ofIL-4,andnoIL-2or interferon-y, when stimulated in vitro with phytohemagglutinin and phorbol myristate acetate. The serum contained eosinophil survival factors whoseactivity was partially neutralized by a specific antihuman IL-5 antibody. This observation further emphasized the relationship between hypereosinophilic syndrome, IL-5, and T-cell lymphoproliferative disorders. 0 1996 by The American Society of Hematology. T s h ~ w n . ' ~Recently, .'~ a case of clonal proliferation of Th2 cells in a man with the hypereosinophilic syndrome (HES) was reported. The investigators showed the expansion of a T-cell clone with the unusual phenotype CD3-CD4' able to produce high levels of L - 4 and IL-5 and low levels of IL2 and IFW-7." We report a similar case of HES in which the presence of a population of CD3-CD4+ cells able to overproduce IL5 (but not IL-4) was shown. HE RELATIONSHIP between hypereosinophilia and T lymphocytes is well established' and is mediated by the production of eosinophilopoietic cytokines. CD4+ lymphocytes are the most important source of granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5, three currently recognized cytokines active in promoting the development and differentiation of eosinophils in bone marrow.z In mice, T-cell clones can be classified according to the pattern of cytokine secretion and there is good evidence that also human cells could be subdivided into these two profile^.^ Type 1 helper T-cell (Thl) clones secrete IL-2 and interferon-y (IFN-y), whereas type 2 (Th2) clones secrete IL-4 and IL-5; both secrete IL-3 and GMCSF. Interestingly, these latter two cytokines also stimulate the development of other marrow-derived cells, whereas IL5 in humans acts more specifically on eosinophils and is the major, and possibly the only, cytokine involved in the production of specific e~sinophilia.'.~ Over the last few years it became evident that the IL-5 production plays a pivotal role in the development of hypereosinophilia both in the secondary forms associated with allergic diseases or parasite infections' and in the idiopathic Interestingly, at least in vitro, these cytokines dramatically increase the life span of eosinophils by inhibiting their apoptotic cell death.'.'' The association between hypereosinophilia and T-cell lymphoma is also well established,""* and in some studies the production of IL-5 by neoplastic T cells has been From the Servizio di lmmunologia Clinica, CONBIOTEC, I I P Laboratorio Analisi, Sezione di Ematologia, Spedali Civili di Brescia, Brescia, Italy; and the Swiss Institut of Allergy and Asthma Research, Davos, Switzerland. Submitted April 21, 1995; accepted September 14, 1995. Supported in part by Consiglio Nazionale delle Ricerche (Grant No. 94.01328.PF39). Address reprint requests to Paolo Airo, MD, Servizio di Immunologia Clinica, Spedali Civili, 25123 Brescia, Italy. The publication costsof this article weredefrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C.section 1734 solely to indicate this fact. 0 1996 by The American Society of Hematology. 0006-4971/96/8704-0043$3.00/0 1416 MATERIALS AND METHODS Case report. A 70-year-old man with hypereosinophilia for the last 5 years has been investigated. His absolute eosinophil count ranged from 2,700 to16,60O/pL.He complained of generalized pruritus with periodic eruptions of papular elements that on histologic examination showed nonspecific follicular inflammation. A physical examination showed mild axillary and inguinal lymph node enlargement and a histologic examination showed a reactive lymphoid hyperplasia with marked infiltration of eosinophils and polyclonal plasmacells with no evidence of lymphoma. Polyclonal hypergammaglobulinemia ( y globulins: 41% of 9.6 g/dL of total serum proteins) was present with high IgG and IgM and normal IgA and IgE levels. Other laboratory parameters were normal as well as peripheral blood eosinophil morphology. Bone marrowbiopsy results showed marked eosinophilia and moderate polyclonal plasmacytosis. Cytogenetic analysis showed a normal karyotype. Causes of secondary eosinophilia were ruled out, including allergic, parasitic, neoplastic, or autoimmune disorders, and a diagnosis of HES was made. Complications of longstanding hypereosinophilia, particularly cardiac, were not found, despite periodic clinical and echographic monitoring. Surprisingly, despite a normal blood lymphocyte count, immunophenotypical studies showed that 38% of lymphocytes were CD3-CD4'. The patient was treated with 6 methylprednisolone (6MP; 16 mg/ d). After 1 month, cutaneous symptoms disappeared and the eosinophil level decreased to 177/pL. He is currently on chronic low-dose 6MP (20 mg/wk). The eosinophil level is in the range of 2,000 to 3,00O/pL. The CD3-CD4' population decreased from 38% to 33% after 1 month and is now 13% to 17%. Cell preparation. Mononuclear cells from the patient and from an age-matched healthy volunteer with no history of allergy and normal lymphocyte populations were separated from the peripheral blood by Ficoll-Hypaque density gradient centrifugation and washed three times in RPMI-1640 (GIBCO Laboratories, Grand Island, NY). Depletion of CD3' cells from peripheral blood mononuclear cells (PBMC) was obtained using negative selection with mouse IgGBlood, Vol 87, No 4 (February 15), 1996: pp 1416-1422 From www.bloodjournal.org by guest on December 22, 2014. For personal use only. HES. IL-5, AND CD3-CD4' T CELLS conjugated magnetic beads (Dynal, Oslo, Norway) precoated for 30 minutes at 4В°C with CD3 monoclonal antibody (MoAb; OKT3; Ortho Diagnostic, Raritan, NJ), as recommended by the manufacturer; less than 4% of negatively selected PBMC expressed CD3 after this procedure, as assessed by immunofluorescence analysis. This population was enriched in CD3-CD4' cells (57%) and was therefore used in experiments of cytokine production as representative of the unusual population. For TCRBV mRNA analysis, CD4TD3cells were further purified from CD3-depleted PBMC by positive selection with CD4-coated beads (Dynal); more than 92% of positively selected cells were CD4+CD3-. For these experiments, CD3+ cells, purified by positive selection (96% CD3+, 61% CD4+, and 36% CDS'), were used as representative of a normal T-cell population. Cell cultures. All experiments were performed in complete medium supplemented with 10% heat-inactivated fetal calf serum (FCS) 1% glutamine, and antibiotics. Proliferative responses were measured in triplicates of 200 pL containing l X lo5 total or CD3depleted PBMC using various combinations of stimulating agents: immobilized anti-CD3 MoAb (60 ng/mL; OKT3); mitogenic combinationof anti-CD2 MoAbs 0KT1lz and OKTll,(l:lOOO; kindly provided by Dr F. Malavasi, University of Turin, Turin, Italy); 1.2 pg/mL phytohemoagglutinin (PHA; Irvine Scientific, Santa Ana, CA), 0.6 pg/mL pokeweed mitogen (PWM; GIBCO); 5 ng/mL phorbo1 myristate acetate (PMA; Sigma Labs, St Louis, MO) plus ionomycin (500 ng/mL; Sigma Labs). After 72 hours of culture at 37В°C. 5% CO,, the cells were pulsed overnight with 1 pCi ('H)-thymidine and were harvested subsequently. Incorporated radioactivity was measured by liquid scintillation counting (Beckman, Fullerton, CA) and the results were expressed as the mean cpm of triplicates. For cytokine production, 5 X lo5 total or CD3-depleted PBMC were cultured at 37"C, 5% CO2 in 24-well flat-bottomed plates in a final volume of 1 mL with or without PHA (1.2 pg/mL) or PHA plus PMA (2 ng/mL). After 72 hours, supernatants were collected and frozen at -20В°C. Immunofluorescence. Evaluation of lymphocyte surface membrane expression was performed on heparinized whole blood samples with the following MoAbs: fluorescein isothiocyanate (FITC)-conjugated OKT3 (CD3), FITC-OKT4 (CD4), FITC-OKT8 (CDS), FITCOKTl1 (CD2), phycoerythrin (PE)-conjugated OKDR (HLA DR), OKMl (CD1 lb) from Ortho, FITC-Leu4 (CD3), FITC-TCR alpha/ beta (TCRAB), PE-Leu 28 (CD28). PE-Leu 45RO (CD45RO), PEIL2R (CD25). PE-Leu1 IC(CD16), Leu 9 (CD7), Leu 7 (CD57), Leu23 (CD69) from Becton Dickinson (Mountain View, CA), FITC-B4 (CD19), PE-2H4 (CD45RA), PE-TI (CD5) from Coulter Immunology (Hialeah, FL), FITC-TCR Delta 1 (TCRGD) from T-cell Sciences (Cambridge, MA), Ber-H2 (CD30) from Dako (Glostrup, Denmark), and 1E9 (CD73; kindly provided by Dr M. Massaia, University of Turin). For unconjugated MoAbs, an indirect immunofluorescence method was applied using an FITC affinity-purified goat antimouse IgG antibody (Ortho). For CD40 ligand (CD40L) and CD70 expression analysis, 2 X IO5 CD3' or CD3-CD4' cells were cultured in 96-well flat-bottomed microtiter plates (Nunc, Roskilde, Denmark) with PMA ( 5 ng/mL) plus ionomycin (500 ng/mL) in a final volume of 200 & / W . After activation (16 hours for CMOL and 96 hours for CD70 optimal expression), cells were harvested, washed two times with phosphate-buffered saline (PBS)-5% FCS, and incubated for 30 minutes at 4"C, respectively, with 50 pL of biotin-conjugated antihuman CD4OL-specific mouse MoAb (TRAP1; kindly provided by Dr R. Kroczek, Robert Koch Institute, Berlin, Germany)2' or 50 pL of biotin-conjugated antihuman CD70 (2F2; kindly donated by Dr R.A.W. Van Lier, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam, The Netherlands); cells 1417 were then washed twice in PBS-FCS and incubated with PE-conjugated Streptavidin (Sigma). After two washes in PBS-FCS, samples were analyzed with a flow cytometer equipped with an argon-ion laser (488 nm; Cytoron Absolute; Ortho) using the ABS software. Gates were selected on lymphoid cells as determined by forward and right-angle scatter properties; dead cells were excluded by setting an appropriate threshold trigger on the low forward light scatter parameter, and nonspecific staining was assessed using FITC-conjugated nonimmune mouse IgG (Coulter). Data were acquired and stored in list mode. Fluorescence analysis was performed on a log scale. Cytokine assays. IL-4 and IFN-y were measured by sandwich enzyme-linked immunosorbent assay (ELISA) as described.22The mouse MoAb 8F1223and biotinylated MoAb 3H4 were used for IL4, MoAb 42-103 (kindly provided by Dr G. Delespesse, University of Montreal, Montreal, Quebec, Canada) and biotinylated KM-48 (Kyowa Medex, Tokyo, Japan) were used for IFN-y determination. The sensitivities of the IL-4 and the IFN-y ELISAs were 30 and 50 pg/mL, respectively. The binding to the second antibody was detected by stepwise incubation with alkaline phosphatase-conjugated streptavidine (Tago 6567; Tago, Burlingame, CA) and 4-nitrophenyl phosphate disodium salt (Merck, Darmstadt, Germany) and detected at 405 nm in a Vmax kinetic microplate ,ELISA reader (Molecular Devices, Sunnyvale, CA). The IL-2 activity was measured by (,H)dThd uptake ofIL-2dependent CTLL cells.z4 Serial dilutions of rIL-2 (1 U biologic activity = 0.1 ng/mL) or IL-2 containing supernatants were incubated with 3 X IO' CTLL cells in 200 pL for 24 hours, followed by a 6-hour pulse of 1 pCi/well of ('H) dThd. This bioassay detects 50 pg/mL of human rIL-2. IL-5 was estimated with a commercial ELISA-Kit (Quantikine; R&D Systems, Minneapolis, MN) as recommended by the manufacturer. Purification of eosinophils. Eosinophils were isolated from human blood by negative selection of granulocytes that do not express CD16 incorporating a magnetic cell separation system (MACS; Miltenyi Biotec, Bergisch-Gladbach, Germany), as previously des ~ r i b e d . The ~ ~ . purity ~ ~ was 98% to 99%as determined by light microscopy and flow cytometry. Cells were washed and resuspended in complete culture medium to a density of 1 X 106/mL. Eosinophil culture. Eosinophils were cultured in complete medium with 5% serum from the HES patient or from a control individual in the presence or absence of an antihuman IL-5 MoAb (IO pg/ mL; R&D Systems, Abingdon, UK) for 48 hours at 3 7 T , 5% COz and 95% air in a humdified atmosphere. Determination of eosinophil death by flow cytometry. The relative amounts of viable cells were determined by uptake of 1 mmoY L ethidium bromide (Becton Dickinson, Mountain View, CA) in dead cells by flow cytometric analysis." DNA analysis. High molecular weight DNA extraction and purification from blood lymphocytes was performed as previously described in detail." DNA wasdigested with three restriction enzymes: EcoRI, HindIII, and BamHI. Restriction fragments were electrophoretically size-separated on gel, transferred to nylon membrane that was prehybridized, and hybridized with '*P-radiolabeledDNA probes. The DNA probe used in T study was genomic probe of the cDNA of the human TCRB chain second constant region gene (CTB, 0.5-kb fragment). Each gel included one lane containing size markers composed of a HindIII digestion of X phage and a lane containing high molecular weight human placenta DNA digested with the same restriction enzymes corresponding to that ofthe other samples in the gel. Hybridization and washings were performed under stringent conditions, and autoradiographic exposures on Kodak X-AR x-ray film (Eastman Kodak, Rochester, NY) were performed for 1 day. RNA extraction and cDNA synthesis and amplification by poly- From www.bloodjournal.org by guest on December 22, 2014. For personal use only. 1418 BRUGNONI ET AL merase chain reaction (PCR). Total RNAwaspreparedfrom Tabb 1. Lymphocyte ImmunophonotypialAnalyrlr CD3'CD4+ and CD3-CD4' T-cell subsets by the guanidiniumthioPatient PBL cyanate-phenol-chloroformmethod.Onemicrogram ofRNAwas Surface Markers 1% positive) Normal Values retrotranscribed in the first strand of the B-chain-specific compleA. Single-color studies mentary DNA (cDNA) using the RiboClone cDNA Synthesis System Kit (Promega Corp, Madison, W)and a primer specific for TCRBCl CD2 78 68-92 and TCRBC2 genes (PcDNA 5' GGG CTG CTC CIT GAG GGG CD5 80 58-85 CTG CGG 3'). A fraction of cDNA was subjected to enzymatic CD7 62 56-84 amplification using a second human TCRBC primer (PA1 5' CCC CD3 40 61-85 ACTGTGCACCTCCTTCC 3') andaTCRBVdegenerated CD4 72 34-54 primer [VPd 5' T(GIT)T(A/C~~)(C~~)T'GGTA(OT)(A/C)(A/G)(A/ CD8 14 15-33 T)CA 3'1 that was designed to amplify B-chain rearrangementsconTCRAB 56-84 31 taining virtually all the known human TCRBV genes?* The condiTCRGD 3 <8 tions generate products of about 430 bp. PCR conditions were perCD16 14 <28 formed under the following conditions: denaturation for 39 cycles CD57 20 4-30 at 93В°C for 1 minute, annealing at 52В°C for 1 minute, and extention CD1 1b 3-33 23 at 72В°C for 1 minute; the last cycle extention was performed at 72В°C CD19 7 2-14 for 7 minutes. The specificity of the total amplified product was HLA-DR 16 2-20 analyzedusingacolorimetricmethodandbiotinylatedTCRBVCD25 7 4 specific probes.29 <l CD40L <l To preventsamplecontaminations,severalprecautionswere CD70 16 <l taken. All solutions were aliqutated and used onlyonce and a negative control (PCR master mix with primersfor TCRBC and TCRBV B. Two-color studies degenerated without cDNAtemplate) was processed with TCR samples in each step of the experiments.In addition, eachT-cell sample CD4tCD338 <l was amplified for the @actin gene to assess the integrity of the CD4'CDZ<l <l mRNA extraction. CD4+CD5<l <l RESULTS Lymphocytesu$acemarkeranalysis. Immunophenotypical studies showed the presence of a CD3-TCR-CD4+ population (Table 1A). All CD4+ cells wereCD2+CD5' andmorethan90%were CD28+CD57-CDllb-CD25DRTD73- (Table 1B). Moreover, all CD4+ lymphocytes present intheCD3-depleted population (ie,CD3-CD4' cells) were CD45RO+ (datanotshown).Unstimulated CD3-CD4+ did not express CD40 ligand or CD30, whereas CD70 was weakly expressed. After stimulation with PMA plus ionomycin, normal expression of CD4OL and unusual higher expression of CD70 compared with CD4+ cells of eight normal controls (85% v 12.1% 2 6.6%) was found on these cells (Fig 1). Proliferative response. Proliferative response of total patient PBMC after activation withPHAand PWM was similar to that of healthy control, whereas it was reduced after activation with CD3 and CD2 (Table 2). On the contrary, CD3-CD4+ enriched PBMC were not able to respond to mitogen activating the cells through cross-linking of membrane receptors (notably PHA, CD3, and CD2), but proliferative response and expression of activation markers such as CD69 and CD25 was partially restored when the transmembrane protein kinase C (PKC) activator PMA was added to PHA or was used in combination with ionomycin. I n vitro cytokine secretion. UnstimulatedPBMCand PBMC depleted ofCD3' cells secrete very low levels of IL-4, L-5, IL-2, and IFN-y, similar to the healthy control (data not shown). As shown in Fig 2, unfractionated PBMC from the patient and healthy control produced similar levels of L-4, L-2, and IFN-y after stimulation with PHA plus PMA. Removal of CD3' cells from patients' PBMC with anti-CD3 MoAb CD4'CD28CD4TD45RA' CD4TD45RO' CD4'DR' CD4'CD25' CD4'CD57+ CD4'CDll 'b CD4'CD30+ CD4+CD73' 5 15 52 3 7 2 3 <l 3 15 25-57 40-72 <7 116 15 <5 <l <l ~ Lymphocyte immunophenotypical analysis. precoated magnetic beads abrogated the ability to produce L - 2 and IFN-y,but did not modify significantly the levels of L - 4 production. Of note, PBMC from patients produced exceedingly high levels of L - 5 on stimulation with PMA plus PHA compared with the healthy control. Also,this production was only partially reduced after removal of CD3+ cells, thus suggesting that this was essentially performed by CD3-CD4+ cells. Serum eosinophil survival factor study.Abioassay of eosinophil in vitro survival showed that patient's serum contained much higher amounts of eosinophil survival factors than that of a healthycontrol, indicating that the CD3-CD4' cells indeed produced cytokines with antiapoptotic properties for eosinophils. A neutralizing antihuman L-5 MoAb partially blocked the effect of the patient's serum (Fig 3). TCR analysis. Southern blot analysis of the DNA extracted from the patient's PBMC showed agermline configuration of the TCRB chain gene with no rearranged clonal bands present on EcoRI-, BamHI-, or HindIII-digested preparations, as in the normal control (Fig 4). TCRBV repertoire analysis performed by reverse transcription-polymerase chain reaction of mRNA extracted from both CD3'and CD3-CD4+ purified T-cell populations showed a polyclonal From www.bloodjournal.org by guest on December 22, 2014. For personal use only. HES,IL-5,ANDCD3-CD4' 1419 T CELLS PATIENT Table 2. Proliferation Studies HEALTHY CONTROL Patient (PBMCI 8 1 " Medium PHA (1.2 mglmL) CD3 (60nglmL) CD2 (1:l.OOO) 18,650 PWM (0.6 mglmL) PMA (5 ng/mL) c ionomycin (500 nglmL) PHA (1.2 mglmL) + PMA (5 nglmL) Patient (PBMC/CD3 ) Healthy Control (PBMC) 770 2 9 7001,230 2 95 72,850 c 1,050 1,050 c 35 115,150 13,300 -c 270 700 -c 58,450 12 2 680 300 2 7 48,550 20,700 c 920 ND 25,400 15,150 ? ND -c 120 59,400 640 5,700 I 140 c 3,200 I 1,600 c 920 c 920 2 1,050 5,400 ND 2 150 Proliferation studies of patients' total and CD3-CD4' enriched PBMC. Data are expressed as the mean cpm 2 SEM of a triplicate culture. Abbreviation: ND, not determined. Fig 1. Expression of CD40L and CD70 on purified CD3-CD4' lymconphocytes of patient(left)and CD3TD4' lymphocytes of normal trol (right). CD4' cells were obtainedby positiveselection with magnetic beads preceded by negative selection with CD3-coated magnetic beads as far as for the patients' cells. The cells were resting or activated with PMA (5 ng/mL) + ionomycin (500 ng/mL). Dashed lines represent the fluorescence of cells stained with nonimmune mouse IgG. Data are presented as relative log fluorescence intensity. that observed is quite frequent in peripheral T-cell lymphoma, being detected in 10% to IS% of In our study, MoAbs directed against two different epitopes of the CD3 molecule were used to exclude the possibility that lack ofrecognition of the CD3 antigen was caused by genetic polymorphism; moreover, the lack of the CD3 molecule is concordant with the lack of reaction with MoAbs directed against the TCRAB and TCRGD frameworks. A further remarkable phenotypic feature of this CD3TD4' lymphocyte population was the expression (constitutively withlow density, strongly positively modulated after stimulation withPMA plus ionomycin) of CD70, a molecule involved in the regulation of T-cell proliferation and survivaL3' CD70 isnot expressed on normal resting CD4' cells and only marginally after stimulation (personal observations). 11-2 IFN-gamma loooo l TCRBV rearrangement in CD3' cells and no RNAtranscription in the CDTCD4' population (Fig 4); in this latter case, the detection of,&actin amplification product showed the integrity of the mRNA extraction and amplification procedure (data not shown). We present a case of HES associated with the presence of a cell population with an unusual CD3-, TCR-, CD4' immunophenotype, without detectable TCRBV mRNA and clonal rearrangement of TCRB gene, which was able to produce large amounts of IL-S after stimulation. This cell population was made of mature T lymphocytes; it had normal lymphocyte morphology and expressed T-cell characteristic antigens (CD2, CDS, CD7, CD28, CD4SR0, and, after in vitro stimulation, CD40L) but not markers (TdT, CDla) of immature T cells. However, these cells lacked surface CD3 and TCR expression. A phenotype similar to 11-5 IL-4 DISCUSSION l4 loooo 1000 O0O0 1 Fig 2. Cytokine production by total PBMC from (0) healthy control and from (m) totaland (a) CD3-depleted (ie, enriched in the CD3-CD4+ population) PBMC from patient in response t o PHA (1.2 pg/mL) PMA (5 ng/mL). + From www.bloodjournal.org by guest on December 22, 2014. For personal use only. BRUGNONI ET AL 1420 Despite its phenotypic abnormalities, therewasno evidence of clonality by Southern analysis of TCRB gene rearrangement. This was performed using three different restriction enzymes, making it unlikely that rearranged bands were missed because of comigration of rearranged DNA fragments.32The detection of germline bands can be explained by the presence of normal non-T cells in the population studied (whole PBMC preparation). Deletion of TCRB gene in an abnormal (clonal?) population would better explain the lack of TCRB gene rearrangement detection as well as the lack of mRNA transcription and of the surface expression of its protein product, which were shown in the present case. Indeed, the absence of clonal TCRB gene rearrangement has been already described in several cases of CD3- peripheral T-cell lymphomas and deletion of the gene has been suggested as a possible explanat i ~ n On . ~ the ~ other hand, it is unlikely that these lymphocytes were of reactive nature. In fact, a stimulus should be hypothesized that is able to induce simultaneously in multiple T-cell clones a failure of TCRBV mRNA transcription and therefore the absence of membrane expression of TCRAB (and CD3). No normal population with these features has been described. We therefore believe that the abnormal lymphocyte population of this patient was the expression of a peripheral T-cell lymphoma with a very indolent clinical course. Interestingly, lymphocytes with the same unusual phenotype were described inpatients in whom the clinical onset of T-cell lymphoma was preceded by a several-year history of eosinophilia''.I3 and ina recently reportedcase of eosinophilia, l 9 1 .c C 0 m C 5 L -e c C 9 E 9 =! .- I5n .- =! CI U C C 'Xc m + m + E E 0 0 2 -g Q, v) U) v) W c c = 8 Fig 3. Effect of sera from the patient withHES or from a normal control individual in the presence or absence of an a n t i - I l l MoAb on thesurvival ofeosinophils. Eosinophils were culturedin complete medium with 5% serum. Viable eosinophils were measured after 48 hours. Data represent mean values of one of three independent experiments. Each experiment was performedin duplicate and also in complete medium with 10% serum, which showed identical results. A 1 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8 B 9 9 1 2 3 4 24 Kb + 11 Kb + 10,sKb + 4Kb 3,s Kb + + 3 RESTR'CT'oN ENZYME Barn HI Eco RI I Hind Ill Fig 4. (A) Southern blot analysis of gene coding for theB chain of theT-cell receptor. DNA from patient PBMC was digested with EcoRl (lane 3). BamHl (lane 6). and Hindlll (lane 9) restriction enzymes and hybridized t o G Bprobe. Each gel included one lane containing size markers composed of a Hindlll digestion of k phage and a lane containing high molecular weight human placenta DNA digested with the same restriction enzymes corresponding t o that of the other samples in the gel. (B) RT-PCR amplification of mRNA from CD3' (lane 1) and CD3-CD4' (lane 2) cells of patient. Lane 3 is the negativePCR control, whereas lane 4 contains synthetic DNA size marker pBR 322 digested Methods, using the human TCRBC primer (PA11and the TCRBV with Hae 111. The amplification was performedas described in the Materials and TCRBV genes. This degenerate primer (Vpd) that wasdesigned t o amplify p chain rearrangements containing virtually all the known human generates products of about 430 bp, as indicated by the arrow. From www.bloodjournal.org by guest on December 22, 2014. For personal use only. HES. 11-5, AND CD3-CD4+ T CELLS in which the abnormal lymphocytes had a typical Th2 cytokine secretion pattern, were clonal, but were not clinically lymphomatous.2oThis case was described as the first example of a proliferation of Th2 lymphocytes in humans. Also in the present case, evaluation of in vitro cytokine production suggested that the abnormal T lymphocytes were responsible for the eosinophilia through the production of IL-5. This was supported by the demonstration of eosinophil survival factors in the patientsвЂ™ serum, whose activity was partially neutralized by an antihuman IL-5 antibody. Their cytokine secretion pattern suggested that they belonged to the Th2 helper lymphocyte subgroup because they did not secrete either IL-2 or IFN-7Unlike . the case reported by Cogan et al,вЂќ our patientвЂ™s cells did not produce high levels of IL-4, in accordance with the low serum IgE level of the patient. This feature did not preclude the assignment of these cells to the Th2 lymphocyte subgroup. Indeed, it has been recognized that the pattern of cytokine secreted by human T-cell clones may be more complex than that of a pure ThlRh2 profile, and it seems that different environmental factors could be responsible for the functional polarization of T-cell clones towards a particular b e h a ~ i o rAlthough .~ common mechanisms in the control expression of IL-4 and IL-5 have been shown, the possibility of a differential regulation has also been reported (eg, IL-2 induces IL-5 but not IL-4 mRNA expression in mouse T cells).33In vivo, a dissociation between IL-4 and IL-5 secretion was reported in nonatopic asthmatic patients.% The lack of TCWCD3 complex may further explain, in our study, this dissociation, as suggested by the ability of cyclosporin to inhibit IL-4 but not IL-5 CD3-induced prod~ction.вЂ™~ Another peculiar feature of this case was the absence of spontaneous in vitro secretion of IL-5, suggesting that persistent cell activation byan as yet unknown stimulus was responsible for its in vivo production. Although the CD3TD4+ lymphocytes were not able to respond to signals acting through the TCFUCD3 complex, they expressed different structures (such as CD2 and CD28) related to the transduction of T-cell activating signals. Studies are in progress to see if the abnormal expression of CD70 by these cells could also play a role in this context. In conclusion, we believe that this is a case of HES pathogenetically linked to the presence of an abnormal population of Th2 helper cells, which was able to selectively secrete large amounts of L - 5 . From a clinical standpoint we will carefully observe the patient because a malignant disease may appear several years after the recognition of eosinophilia.ll.вЂ™3-15Moreover, because in this case the absolute lymphocyte count was within normal range, we suggest that a lymphocyte immunophenotypic analysis should be included in the diagnostic workup of every case of HES to detect the presence of even small subpopulations withan abnormal phenotype. Future studies should determine whether there is a specific association between eosinophilia and the CD3-CD4+ phenotype of lymphocytes, as suggested by this and several other reports.вЂ™1,13*20 REFERENCES 1. Basten A, Beeson PB: Mechanism of eosinophilia. 11. Role of the lymphocyte. J Exp Med 131:1288, 1970 1421 2. Weller PF, Bubley GJ: The idiopathic hypereosinophilic syndrome. Blood 83:2759, 1994 3. Romagnani S: Human Thl and Th2 subsets: Doubt no more. Immunol Today 12:256, 1991 4. Sanderson CJ: Interleukin-5, eosinophils and disease. Blood 79:3101, 1992 5. Romagnani S: Lymphokine production by human T cells in disease states. AMU Rev Immunol 12:227, 1994 6. Owen W, Rothenberg ME, Petersen J, Weller PF, Silberstein D, Sheffer AL, Stevens RL, Soberman RJ, Austen KF: Interleukin 5 and phenotypically altered eosinophils in the blood of patients with the idiopathic hypereosinophilic syndrome. J Exp Med 170:343, 1989 7. 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For personal use only. 1996 87: 1416-1422 A case of hypereosinophilic syndrome is associated with the expansion of a CD3-CD4+ T-cell population able to secrete large amounts of interleukin-5 D Brugnoni, P Airo, G Rossi, A Bettinardi, HU Simon, L Garza, C Tosoni, R Cattaneo, K Blaser and A Tucci Updated information and services can be found at: http://www.bloodjournal.org/content/87/4/1416.full.html Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. 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