Vox Sang 1991;60:189-190 0 1991 S. Karger AG, Basel

0042-9007/9Y0603-0189 $2.75/0

T Lymphocyte Colony Assay in Patients with Idiopathic Autoimmune Hemolytic Anemia: Preliminary Results

Roberto Conte, Cristina Tassi, Andrea Bontadini, Daniela Belletti, Pier Luigi Tattari Servizio di Immunoematologia e Trasfusionale, Policlinico S. Orsola; Istituto di Ematologia L. e A. Seragnoli deU’Universitk, Bologna; Istituto Nazionale Ricerca sul Cancro, Genova, Italia

Several cellular immune abnormalities have been found in patients with idiopathic autoimmune hemolytic anemia (IAHA). To date, decreased Tcell number, imbalanced T cell subsets, reduced response to phytohemagglutinin, impaired autologous mixed lymphocyte reactions, and a functional deficiency of NK cells have been reported in these patients [1-4].

It has been suggested that the T lymphocyte colony assay is a highly sensitive test capable of detecting ab- normalities not revealed by other parameters of cellular immunity [5-71. Thus, we have performed a preliminary study regarding the T lymphocyte colony assay in 2 ‘warm type’ IAHA patients to further investigate disorders of the T-cell compartment in this disease.

Patient 1 (male, 75 years old) had a strongly positive DAT, while patients 2 (female, 63 years old) was in serolog- ical remission. Both patients were in hematological remis- sion and off treatment for at least 6 months.

We have employed a culture method previously report- ed [3, 8, 91, with slight modifications to induce the clonal proliferation of T lymphocytes. Briefly, mononuclear cells isolated by Ficoll-Hypaque gradient density centrifugation were divided in two aliquots. One aliquot was suspended in RPMI 1640, irradiated with 20 Gy, and used as feeder layer. The second aliquot was suspended in RPMI 1640 supple-

Table 1. Cloning assay in IAHA patients

mented with antibiotics, L-glutamine, 10% pooled AB se- rum, 200 U/ml recombinant IL-2, and 30% (v/v) culture supernatant derived from the UCHTl hybridoma (CD3 monoclonal antibody, kindly supplied by Prof. P.C.L. Be- verly, ICRF, London, UK). Cells were then cloned by limit- ing dilution (0.5-5-1-10 cells/well), three times at least, on an autologous feeder layer. The culture medium was re- placed with fresh medium every 7 day. A phenotype analy- sis was carried out by flow cytometer (FACStar, Becton Dickinson) at the beginning of the culture period and at the second, sixth, and ninth week of culture using a panel of monoclonal antibodies specific for CD3, CD4, CD8, CD25, DR, CD16, CD57, and TCRy/61 antigens (Becton Dickinson). In each experiment, the clonal efficiency of Tcells was evaluated comparing the number of clones ob- tained in every plate from the patient with the number of clones obtained from a normal donor following the same culture procedure.

The results are reported in tablel. The patient with serological markers of active disease (No. 1) showed a very low cloning efficiency in comparison to that of the control, while the patient in serological remission (No. 2) showed a cloning efficiency close to that of the control. As expected, a significant predominance of CD4+ clones was found in patient 1 (87%).

Patient DAT test Pre-culture phenotype, % Clonal efficiency Clone phenotype, % No patient: control

type score CD3+ CD4+ CD8+ CD4+ CD8+

4+ 90 85 2 25: loo 87 13 70 58 19 79: 100 65 35 -

1 IgG 2 -

190 Conte/TassBontadini/Belletti/Tazzari

Table 2. Cloning assay: phenotypes 2 Conte R, Dinota A, Tauari PL, et al: Analysis of natural killer cells in patients with idiopathic autoimmune hemolytic anemia. Vox

3 Kruger J, Rahman A, Mogk KU, et al: Tcell deficiency in patients CD25 CD57 CD16 DR TCRy/Sl with autoimmune hemolytic anemia (‘warm type’). Vox Sang

1 CD4+ 52 62 0 9 0 0 4 Reinherz EL, Rubinstein A, Geha RS, et al: Abnormalities of CD8+ 25 100 0 9 0 0 immunoregulatory T-cells in disorders of immune functions. N Engl

2 CD4+ 48 0 0 85 0 5 Bernstein ML, Winkelstein A, Dobson SA: Depressed T cell colo- CD8+ 22 0 0 loo 0 ny growth in systemic lupus erythematosus: Arthritis Rheum 1980;

6 Ragni MV, Winkelstein A, Evans TL, et al: T lymphocyte colony

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Clone phenotype analysis is shown in table 2. Again, a significant difference concerning clone phenotypes was found between the two patients. A peculiar phenotype, characterized by the concomitant expression of both CD4 and CD57 antigens was found only in patient 1. This pheno- type is usually found only in subsets of lymph node CD4- positive cells [lo]. Thus, it would be of interest to further investigate whether CD4/CD57-positive cells might play a role in autoimmune disorders, particularly in IAHA.

These clones have been submitted to complete pheno- type analysis. Furthermore, a study on the possible lytic function of this particular subclass of Tcells is in progress.

These preliminary data seem to confirm and extend our previous observations on the presence of cellular immun- ologic abnormalities, which are involved in the appearance and development of the autoimmune disorder in IAHA patients.

7 Winkelstein A: Dependency of human T lymphocyte colony forma- tion on soluble factors produced by accessory or tumor cells. J Immunol1983;130:2715-2719.

8 Janssen 0, Nerl C, Kabelitz D: Tcells in B-cell chronic lymphocytic leukemia: Quantitative assessment of cytotoxic and interleukin 2-producing lymphocyte precursors by limiting dilution analysis.

9 Kabelitz D, Jansen 0, Brucker C: Use of OKT3 hybridoma cells to clonally activate CD3+ human T lymphocytes. J Immunol Methods

10 Janossy G, Amlot G: Immunofluorescence and immunohistoche- mistry; in Klaus GGB (ed): Lymphocytes - A Practical Approach. Oxford, IRC Press, 1989, pp 67-108.

11 Weber WEJ, Buurman WA, Vandermeeren MMPP, et al: Activa- tion through CD3 molecule leads to clonal expansion of all human peripheral blood T lymphocytes: Functional analysis of clonal ex- panded cells. J Immunol1985;135:2337-2341.

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References Dr. Roberto Conte Servizio di Immunoematologia e Trasfusionale Policlinico S. Orsola Via Massarenti 9 I 4 1 3 8 Bologna (Italy)

1 Conte R, Tazzari PL, Finelli C: Deficiency of autologous mixed lymphocyte reaction in patients with idiopathic autoimmune hemo- lytic anemia. Vox Sang 1985;49:285-291.