Haematologica 2000; 85:118-123 Phagocytes & Lymphocytes original paper Analysis of T-cell clones in systemic lupus erythematosus MARCELLA CONVERSO, MARIA TIZIANA BERTERO, ANTONELLA VALLARIO, FEDERICO CALIGARIS-CAPPIO Dipartimento di Scienze Biomediche e Oncologia Umana, Università di Torino, Ospedale Mauriziano Umberto I°, Turin and Istituto per la Ricerca e la Cura del Cancro (IRCC), Candiolo, Italy ABSTRACT Background and Objectives. It is fairly well estab- nificant insight into normal T-cell function, it is now being evaluated whether pathologic conditions may lished that T-helper (TH)1 cells play a role in the pathogenesis of organ-specific autoimmune dis- be influenced by one of the two alternative polarized 2 eases, while their role and their relationship with responses. It is fairly well established that TH1 cells TH2 cells is far from being defined in systemic lupus play a role in the pathogenesis of organ-specific 1,4 erythematosus (SLE). To address this issue, six autoimmune diseases, while their role, as well as female patients who fulfilled the American Rheuma- their relationship with TH2 cells, in systemic autoim- 1 tism Association criteria for the diagnosis of SLE mune diseases is far from defined. This is especial- were studied. ly true in systemic lupus erythematosus (SLE), a mul- tifactorial systemic autoimmune disease character- Design and Methods. We analyzed the intracellular ized by abnormal immune function with increased production of cytokines by T-cells from the periph- numbers of hyperactive B-cells together with eral blood (PB). Then, we established T-cell clones impaired T-cell regulation and a dysfunction of anti- (TCC) from the peripheral blood (PB) of all cases as gen-presenting cells.5 A number of observations in well as from the synovial fluid of one patient with an murine SLE models suggest a role for TH2 cells. In articular flare-up. NZB.H-2bm12 mice all TH cell clones able to provide Results. The percentages of IL-4 positive and IFN-␥ help for anti-DNA production secrete IL-4, but not positive PB T-cells were not different between SLE IFN␥6 and the treatment of NZB/NZW mice with patients and normal controls. When 93 TCC (67 antibodies that block the interaction between B and CD4+, 23 CD8+) from the PB of 5 different SLE TH2 cells significantly ameliorates the SLE-like dis- patients were compared to 118 TCC (94 CD4+, 23 ease.7 However, another set of observations indicate CD8+) from 5 healthy controls no statistical differ- that TH1 cells may be significantly involved. Not only ence was observed between SLE and controls in IL-4, but also IL-12 is important in the genesis of SLE 8 terms of TH1, TH2 or TH0 phenotype. However, SLE in NZB/WF1 mice and IL-4 protects NZW x clones showed a reduced ability to secrete IL-10 C57BL/6. Yaa mice from the development of a lethal (p = 0.002). In contrast, the analysis of the 30 genetically linked lupus-like glomerulonephritis.9 clones obtained from synovial fluid revealed that Also, a study of the kinetics of cytokine production + 11/23 CD4 clones were TH1, 12/23 were TH0, 2/7 in experimental SLE has revealed that TH1 type or + CD8 clones were TH1 and 5/7 were TH0. No TH2 TH2 type cytokines predominate in different phases clones were obtained from the synovial fluid. of the course of the disease.10 To elucidate which T-cells play a role in human Interpretation and Conclusions. The data suggest SLE and through which cytokines, we studied the T that the T-cell subsets operating in actively inflamed cell intracellular production of cytokines and ana- organs of SLE may belong to the TH1 and TH0 sub- lyzed T-cell clones (TCC) generated from patients sets. with SLE. ©2000, Ferrata Storti Foundation Design and Methods Key words: systemic lupus erythematosus, autoimmune diseases, T helper (TH) lymphocytes, cytokines, IL-10 Patients Six female patients, aged 18-43 years, who fulfilled the American Rheumatism Association criteria11 for unctionally polarized responses can be detected the diagnosis of SLE, were studied. within the CD4+ T helper (TH) lymphocyte sub- 1-3 First, the intracellular cytokine profile was evaluat- sets by the pattern of cytokines produced. TH1 F ␥ ␥ ed in peripheral blood lymphocytes (PBL) from cells essentially produce interferon- (IFN ); TH2 patients (and from 7 controls matched for age and cells produce interleukin 4 (IL-4), IL-5, and IL-10. T- sex). Next, T-cell lines (TCL) and T-cell clones (TCC) cells with a mixed pattern of cytokine production were generated from the PB of 5 patients (and 5 have been designated as TH0. Since the investigation healthy volunteers, matched for age and sex, used as of different TH cell populations has provided a sig- controls). In one patient, who had a flare of disease activity characterized by non-erosive gonarthritis, TCC Correspondence: F. Caligaris-Cappio, M.D., Ospedale Mauriziano were also generated from the synovial fluid (SF). On Umberto I°, largo Turati 62, 10128 Turin, Italy. Phone: international +39-011-5080650 – Fax: international +39-011-5682588 – E-mail: the day of sampling, these patients’ history and cur- [email protected] rent treatment were recorded and disease activity Haematologica vol. 85(2):February 2000 T-cell clones in SLE 119 assessed according to the European Consensus Study stored in listmode data files. Each measurement Group (ECLAM) scoring system.12 Two patients had acquired 20,000 cells. not yet started any therapy, one was receiving low dose steroids, one low dose steroids plus antimalarials and Generation of T-cell lines and clones one low dose steroids, antimalarials and azathioprine. Mononuclear cells obtained from the PB of 5 patients and 5 healthy volunteers and from the syn- Reagents ovial fluid of 1 patient were resuspended in RPMI Phytohemoagglutinin (PHA) was purchased from 1640 medium supplemented with 2 mM L-gluta- Gibco BRL (Grand Island, NY, USA), phorbol myristic mine, 2ϫ10-5 M 2ME and 10% heat-inactivated FCS acid (PMA), ionomycin and brefeldin from Sigma (complete medium) at the concentration of 106/2 Chemical Co. (St. Louis, MO, USA). Human recom- mL. To generate TCL, mononuclear cells were stim- binant IL-2 was a kind gift from Eurocetus (Milan, ulated in 24-well plates for 10 days in the presence of Italy). OKT3 (anti-CD3) monoclonal antibody (MAb) human IL-2 (50 U/mL) and PHA 1‰ in an atmos- was purchased from Ortho Pharmaceuticals (Raritan, phere with 5% CO2 in air. NJ, USA). To generate TCC, T-cell blasts obtained from TCL The monoclonal antibodies (MAbs) used for flow were seeded under limiting dilution conditions (1 cell cytometric analysis were CD3-FITC (Leu 4, Becton per well) in 10 round-bottomed microwell plates con- Dickinson, cat. n. 92-0001), HLA-DR-PE (Becton Dick- taining 105 irradiated PBMC (6,000 rad) as feeder cells inson, cat. n. 7367), CD4-Tri-color (Caltag, San Fran- and PHA (0.5% vol/vol) in a final volume of 0.2 mL of cisco, CA, cat. n. MHCD0406), CD8-Tri-color (Caltag, complete medium supplemented with IL-2 (70 U/mL) cat. n. MHCD0806), IL-4-PE (Becton Dickinson, cat. and 10% FCS (Hyclone Laboratories Inc.). n. 340451), ␥IFN-FITC (Becton Dickinson, cat. n. Growing microcultures were then supplemented, 340449). at weekly intervals, with IL-2 (50 U/mL) and 105 irra- Cell separation PBL were obtained by separation diated feeder cells. on Ficoll-Hypaque (FH; Pharmacia-LKB, Uppsala, Sweden) gradient and washed twice in phosphate Assessment of cytokine secretion profile of TCC buffered saline (PBS). Cells from synovial fluid were The cytokine secretion profile of T-cell clones was collected by centrifugation after PBS dilution. evaluated by stimulating 106 T-cell blasts from each clone in 1 mL of complete medium with PMA (10 Intracellular cytokine analysis ng/mL) plus anti-CD3 MAb (100 ng/mL) to achieve Cells were cultured in 24-well plates (Celbio, Milan, maximal stimulation. After 36 h stimulation, the cul- Italy) at a concentration of 2ϫ106/mL for 10 hours ture supernatants were collected and stored at –80°C at 37°C in an humidified atmosphere containing 5% until used. CO2 in RPMI 1640 medium supplemented with 10% Supernatants were assayed for IFN␥, IL-4, IL-5 and fetal calf serum (FCS). Cells were then stimulated IL-10 content. The quantitative determinations of with PMA (30 ng/mL), ionomycin (1 µg/mL) and IFN␥, IL-4, IL-5 and IL-10 were performed by com- brefeldin (10 µg/mL) for 4 hours before the end of mercial ELISA (Benfer-Scheller, Keystone Laborato- culture. Cultured cells were washed twice in PBS, ries, CA, USA) according to the manufacturer’s stained with CD4 and CD8 MAb for 15 min at room instructions. Values of cytokine content 5 standard temperature and washed with PBS. The cells were deviations (SD) greater then those detected in con- then fixed and permeabilized with FACS permeabi- trol supernatants (obtained by stimulation of irradi- lizing solution (Becton Dickinson, cat n. 340457) ated feeder cells alone) were regarded as positive. The according to the manufacturer’s instructions. Fixed following values were considered positive: IFN␥ >0.8 cells were washed with PBS containing 1% bovine ng/mL, IL-4 > 150 pg/mL, IL-5 > 400 pg/mL, IL-10 serum albumin (BSA, Sigma, St. Louis, MO, USA) > 200 pg/mL. and 0.1% sodium azide and then reacted with MAb Clones producing IFN␥ but not IL-4 and/or IL-5 ␥ to IL-4 and IFN for 30 min at room temperature. were classified as TH1-like, clones producing both ␥ Flow cytometric analysis IFN and IL-4 and/or IL-5 were classified as TH0-like, and clones producing IL-4 and/or IL-5 but not IFN␥ An aliquot of cells was resuspended in PBS con- were classified as TH2-like.