cell culture supernatant were included instead of the patient's cells in NR cultures. ... Suppressor T cells have also been implicated in the regulation of T cell ...
AJEBAK 59 (Pt. 3) 263-275 (1981)
© SUPPRESSOR CELL ACTIVITY IN A PROLIFERATIVE DISORDER OF T LYMPHOCYTES by ANN KUPA^ MIRIAM E. THOMAS*, HELEN MOORE* J. BRADLEY*, H. ZOLA*, M. HOOPERf AND P. HARDlNGf (From the 'Department of Clinical Immunology, Flinders Medical Centre, Bedford Park, South Australia 5042, and the fRoyal Adelaide Hospital. Adelaide, South Australia 5000.) (Accepted for publication January 12,1981.) Summary. We report details of the immunological profile of a patient with the candidiasis endocrinopathy syndrome who h:is developed T-type chronic lymphocytic leukaemia. The patient is anergic to a panel of deiiiyed hypersensitivity skin tests, and has poor in vitro mitogenic responses, but B cell function rn vivo is not impaired. Subsequent functional studies have revealed that cells from the patient have a significant suppressive efTect in coculture (p < 0.05) on the responses of healthy donor lymphocytes (NR) to the mitogen phytohaemaggtutinin (PHA). A degree of selectivity for the suppressive efFect is suggested by the lack of similar efFects on coculture responses to the milogens cocanavalin A (Con A) and pokeweed mitogen (PWM ). Milomycin C treatment of the patient's cells reduced their suppressive activity but significant suppression was still observed in the majority of PHA cocultures. The suppressor activity required the presence of the patient's cells in cocultures, as no suppression was observed when the patient's serum or cell culture supernatant were included instead of the patient's cells in NR cultures.
INTRODUCTION It is generally accepted that certain subpopulations of T cells play an important regulatory role in immunologic responsiveness. Initial studies in mice and humans focused on regulation of B cell responses by helper or suppressor T cells. Suppressor T cells have also been implicated in the regulation of T cell functions such as delayed hypersensitivity and tumour immunity. Whisler and Stobo (1978) noted that distinct T cell subpopulations were responsible for suppression of humoral and delayed hypersensitivity responses. There are considerable experimental dilTiculties in studying regulatory T cell subsets in man. Methods of study include mitogen activation of suppressor T cells (Shou, Schwartz and Good, 1976) or activation of nonspecific or specific suppressor cells in mixed lymphocyte reactions (MLR) (Hirschberg and Thorsby, 1977; Haynes, van Speybroeck and Cochrum, 1978). This type of experiment is complicated by the potential activation of opposing regulatory subsets. Recently, emphasis has been placed on the separation of
264
KUPA, THOMAS. MOORE. BRADLEY, ZOLA, HOOPER AND HARDING
T cell subsets on the basis either of subset specific antisera or of the differential expression of Fc receptors. Patients with proliferative disorders of lymphocyte subpopulations provide material for functional studies which may be relevant to the functional interactions of normal cells. Thus, Broder et al. (1976) have demonstrated the ability of Sezary cells to provide T cell help for immunoglobulin synthesis. There has also been a report of T-type acute lymphoblastic leukaemia cells capable of producing helper activity for B lymphocyte differentiation (Chiao etai, 1979). We report in this paper studies of suppressor cell activity in a proliferative disorder of T lymphocytes occurring in a patient with the candidiasis endocrinopathy (CE) syndrome—a condition in which multiple endocrine disorders due to autoimmune processes are associated with a defect in cell mediated immunity which renders the patient susceptible to candidal infection of skin and mucous membranes. Case report The patient is a 40-year-old housewife with idiopathic hypoparathyroidism, Addison's disease and primary ovarian failure. Severe candidal infection of nails, mouth and oesophagus have been a problem since early childhood. Delayed hypersensitivity skin testing to Candida is negative. In 1968 lymphocytosis was noted on peripheral blood picture, and in 1975 bone marrow biopsy revealed 60% of marrow cells to be mature lymphocytes, consistent with a diagnosis of chronic lymphocytic leukaemia (CLL). The patient was asymptomatic, and clinical examination revealed only mild hepatomegaly and small axillary and groin nodes. In 1976, an episode of Coombs-positive haemolytic anaemia responded to a course of chlorambucil atid prednisolone. Antinuclear factor has been present in high titre since 1975, but there have been no clinical features to suggest a diagnosis of systemic lupus erythematosis (SLE) with the possible exception of the haemolytic anaemia. In late 1976 lymphocyte surface marker studies suggested that the majority of lymphocytes were T cells. This finding was confirmed in May, 1979 (Table 1) and it was noted that 45% of the patient's lymphocytes bore receptors for the Fc portion of IgG. As suppressor T cells are found within the Fcy subset, an experiment was devised to assess possible modulatory effects of the patient's cells on responses of normal lymphocytes to the mitogens PHA, PWM and Con A. The patient's own mitogenic responses were low, suggesting either that the patient's cells were incapable of responding or that the responses were being actively suppressed. MATERIALS AND METHODS Cell preparation and characterization All cell studies were performed on mononuclear cells separated from heparinized peripheral blood on a Ficoll-Hypaque gradienl. These ceils are referred to as PBL and, in the case of the patient J, the monocyte count would have been very low because lymphocytes comprised 98% of total white cell count.
265
SUPPRESSOR ACTIVITY IN T TYPE C.L.L. TABLE 1 Lymphocyte surface markers. May, 1979 White cell count: 38.700/*l 98% lymphocytes Total lymphocytes: 37,926/^1 % PBL
B CELLS Surface membrane Ig Surface membrane IgM Complement C3 receptor Mouse RBC rosette Fc receptor Monoclonal anti B (FMCl) r CELLS SRBC rosette Anti T cell serum MONOCLONAL ANTI la" (FMC4)
% Marrow
Patient
Reference range Mean ± 2SD
Patient
1 2 12 8 45 4
(5-19) ( 3 - 9) (7-16) (8-24) (7-30) (control : 8)
2 ND 7 6 40 2
93 82 8-11%
(64-84) (50-80) (15-21)
87 80 ND
*This monoclonal antiserum was not used in May, 1979. Results quoted arc those from subsequent studies in October, 1979, and February, 1980. Lymphocyte marker assays Details of the procedures used in this laboratory have been described previously (Brooks el al. 1980; Beckman et al, 1980). T lymphocytes were identified by roselting with papaintreated sheep erythrocytes and by indirect immunoiluorescence using an antiserum specific for human T lymphocytes (HTLA, heterologous antiserum. Zola and Valdimarsson, 1978). Cells with surface membrane immunoglobulin were detected by direct fluorescence using rabbit anti-human !g (polyvalent) (Wellcome Reagents, Beckenham, Kent) and anti-IgM (Behringwerke AG, West Germany). Cells bearing receptors for complement components were enumerated by rosette formation with complement coated zymosan particles. Cells with receptors for the Fc portion of IgG were assayed by rosette formation wiih ox red blood cells sensilized wiih rabbit anti-ox IgG antibodies. Rosetting with papain-treated mouse erythrocytes was used as an additional marker for B and null cells. B cells and la-posilive cells were detected using the monoclonal antibodies FMCl (Brooks et al, 1980), and FMC4 (Beckman et al. 1980). Lymphocyte stimulation studies Standard transformations. PBL from palient J and two normal donors were cultured at a concentration of 0 5 x !0« cells/ml, in wells of microplates (Linbro. Flow Labs Inc.. tissue culture plates—96 round-bottom wells) in RPMI 1640 medium supplemented with glutamine (2mM), penicillin (100 iu/ml) and streptomycin (100 iu/ml) and 20% pooled ABO serum (heat-inactivaled). The mitogens purified PHA (Wellcome Reagents), Con A (Calbiochem) or PWM (Sigma Chemical Co.) were added to the triplicate wells at 3 concentrations of each mitogen: PHA 8 16 32 /"g/ml of culture Con A 20 40 160 Mg/ml of culture PWM 00125 0 25 2 5 Mg/ml of culture The cultures were incubated at 37° in a humidified atmosphere with 5% CO2 for 68 h, then pulsed for 4 h wiih 0 4 ^Ci triliated thymidine per well (6--H-thymidine aqueous solution 5ci/mmol. the Radiochemical Cetitre, Amersham, U.K.) and harvested on a Skatron multi-harvester (Flow systems) on to glass fibre filter paper. After drying, the glass
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KUPA, THOMAS, MOORE, BRADLEY, ZOLA, HOOPER AND HARDING
fibre discs were placed in scintillation fluid (scintillation grade toluene containing 3 91 g/1 PPO and 0 39 g/I POPOP) and ^H emissions counted on a liquid scintillation counter. Results were e.xpressed as the mean of triplicate cultures in c.p.m.. plotted on a logarithmic scale and compared with a previously determined reference range. Suppressor cell activity. Cells from the patient J and from a normal control (NC) were assessed for their ability to modulate the mitogenic responses of cells from a second normal donor (NR). Cocultures were set up using the normal responder (NR) cells mixed with the putative modulator cells in varying ratios: 75:25, 50:50, 25:75. The total cell number and concentration in the cocultures was kept constant at 1 x lO""' cells in 200 M1. PHA. Con A and PWM were added to triplicate wells as above. Controls included wells with only NR cells at varying concentrations, corresponding to the number of NR cells in the cocultures. These were designated NRyr.. NR:,o and NR-^,-. and they assessed ihe effects of simple dilution on mitogen responsiveness. The culture volume was not altered. NC and I cells were treated with mitomycin C (25 ^g/ml for 30 min) to eliminate DNA synthesis, and the mitomycin-lrealed cells, designated Ncf" and J"', were used in parallel coculture experiments. The efficacy of mitomycin C treatment was confirmed by a reduction of more than 97% in the PHA responses of NC cells. A.'isessment of soluble suppressor factors. Further lymphocyte transformation studies were carried out using NR cells, according to the standard method described above, but with substitution of the pooled ABO serum by J serum, or with substitution of varying proportions of the RPMI 1640/ABO serum by supernatant from a 3-day unstimulated culture of J cells. Humoral immunity Serum immunoglobulin levels were determined by automated nephelometry (Technicon, A.LP.). Candida preclpitins were detected by the Ouchterlony double difTusion technique. Specific IgM and IgG anlibody responses to typhoid immunization ( 0 1 ml intramuscular injection of typhoid vaccine. C.S.L. Auslralia) were assessed by comparing Salmonella typhi O and H liires prior to immunization with those 14 days after immunization. Isoagglutinin levels were determined by direct haemagglutinin at 4° for 30 min. Autoantibodies were detected by standard indirect immunoduorescent technique. Delayed hypersensilivity responses were estimated by measurement of area of induration 48 h after intra-epidermal injection of 0-1 ml of the following antigens: Streptokinase/streptodornase (SK/SD) 6 25 units Lederle Labs, Australia Tuberculin purified protein 10 units derivative (P.P.D.) C.S.L.. Australia Candida albieans—Allergic extract l/IO and 1/100 Dermatophytin 'O' Hollister Stier Lab. dilutions of extract .Statistical analysis of results Logarithmic transformation of PHA dose-response curves has been shown to approximate to a normal distribution (Ziegler et al. 1974). For each concentration of mitogen, comparisons between the incorporated radioactivity of the triplicate cultures were conducted using analysis of variance of logarithmically transformed data.
RESULTS Standard transformations Mitogenic responses of NR cells to PHA, PWM and Con A were normal, while mitogenic responses of J cells to the three mitogens were low, especially to PHA (Fig. 1).
SUPPRESSOR ACTIVITY IN T TYPE C.L.L. PHA Transformation
16
Fig. I.
PWM Transformation
Con A Transformation
^0-25 2-5 20 40 0-0125 Mitogen concentration pg/ml culture 32
267
160
Standard transformations.
Reference range mean ± 2 S.D.
NR cells
- J cells
Co-culture responses (The statistical significance of the data in Figs. 2, 3, 4, 5 and Table 2 is presented in Table 3.) PHA (Table 2, Figs. 2 and 3) The addition of mitomycin C-treated NC cells (NC*") to NR cells produced no significant fall in NR responses (p > 05). Results were comparable to those of control cultures NRT:,, NR-,O, and NR:;.-,, where the progressive fall in counts refiected dilution of NR cells (Fig. 2). The addition of untreated NC cells to NR cells resulted in a marked increase in thymidine incorporation at all cell ratios (Fig. 3), consistent with additive NR and NC responses to PHA and to allogeneie cells (an early, twoway MLR). In contrast, the addition of J or J"" cells to NR cells resulted in a reduction of PHA responses of NR ceils at all cell ratios tested (Fig. 2) and at all concentrations of PHA (Fig. 3). Con A (Table 2) There was no consistent pattern of responses when J or J"" cells were added to NR cells. In fact, at cell ratio NR:J 25:75 there was a significant increase in counts. PWM (Table 2) There was no consistent reduction in tritiated thymidine uptake by NR cells following the addition of J or J'" cells. The cultures with the lowest levels
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KUPA, THOMAS, MOORE. BRADLEY, ZOLA, HOOPER AND HARDING
25000 -
20000-
B 3 •i
15000-
0)
PHA 32>jg/ml
a ?Z3 10000-
o O
5000-
i=0 NR 1001 50
0
75 25
% Responder cells
100 % Modulator cells
Fig. 2. Comparison of NR and coculture responses to PHA at varying cell ratios. Verticlar bars represent range of triplicate results. D NR cells alone
Modulator cells added
0 of PWM showed increased thymidine uptake when cocultured with J and J™, but, since cocultures with control modulator cells (NC") showed greater increases, the effect cannot be ascribed to J cells. Soluble suppressor factors Substitution of J serum for pooled ABO serum did not affect NR responses to PHA. Con A or PWM. Substitution of supernatant from J cell culture for varying proportions of the standard medium did not affect NR mitogenic responses.
Humoral immunity (Table 4) Lnmunoglobulin levels, serum electrophoresis, isoagglutinin levels and typhoid immunization responses were all normal. Strong Candida precipitins were detected. Autoantibody screens revealed a titre of anti-nuclear antibody (ANF) of 1/1280. with positive DNA binding by the Crithidia assay, and anti-adrenal antibody, titre 1/20. Delayed hypersensitivity tests Responses to all skin tests including the Candida skin tests were negative.
SUPPRESSOR ACTIVITY IN T TYPE C.L.L.
269
,28152
22000• 20000-
18000•
16000•
14000•
B I 12000 a. « 10000 c O 8000-
6000
4000
2000
Control NR50
Nc Modulator cells added to NR celts-Ratio 50:50
Fig. 3. PHA responses of NR cells—alone and in coculture, cell ratio 50:50. Vertical bars represent range of triplicate results. * These values represent mean of duplicate results. Third culture result not included as count
