Int J Clin Lab Res 21: 186-189, 1991 S CH 9Springer-Verlag 1991 Cytokine production by allergen (DerpI)-specific CD4 + T cell clones derived from a patient with severe atopic disease Paola Parronchi, Roberto Manetti, Cecilia Simonelli, Francesco Santoni Rugiu, Marie-Pierre Piccinni, Enrico Maggi, and Sergio Romagnani Immunologia Clinica e Allergologia, Istituto di Clinica Medica 3, Universitfi degli Studi di Firenze, Viale Morgagni 85, 1-50134 Florence, Italy Summary. Twenty-four T cell clones (TCC) specific for purified Dermatophagoides pteronyssinus group I aller- gen (Der p I) were established from the peripheral blood of a patient with severe atopic disease and assessed for cytokine production in response to stimulation with both phorbol 12-myristate 13-acetate and anti-CD3 mono- clonal antibody. All Der p I-specific TCC produced high amounts of interleukin (IL)-4 in association with IL-3, IL-5 and granulocyte monocyte-colony stimulating fac- tor (GM-CSF), whereas they produced variable amounts of IL-2 and virtually no interferon-~:. These data support the hypothesis that atopy is associated with preferential activation of type 2 T helper ceils and suggest a deregula- tion in the function of the IL-3, IL-4, IL-5 and GM-CSF gene cluster in subjects with severe atopic disorders. Key words: Atopy - T cell clones - Interleukin-2, -3, -4 and -5 - Interferon-y - Granulocyte monocyte-colony stimulating factor Introduction The release of histamine, leukotrienes and other media- tors in response to the cross-linkage of receptors for the Fc region of IgE (FeaR) on the surface of mast cells and basophils is responsible for allergic symptoms. The FceR cross-linkage is usually induced by the interaction be- tween cell-bound IgE antibody and the specific allergen. The hallmark of allergic (atopic) patients is, indeed, the ability of their B cells to produce IgE antibody in re- sponse to common environmental allergens. Recently, the regulatory mechanisms of IgE synthesis have been clarified. Interleukin (IL)-4 produced by T helper (T.) cells is responsible for the induction of IgE synthesis by B cells, whereas interferon (IFN)-y, another TM cell- derived cytokine, exerts a negative regulatory effect (re- viewed in [8]). Offprint requests to: S. Romagnani In previous papers, we [6] and others [10] have shown that the great majority of human CD4 § T cell clones (TCC) established from atopic individuals are able to produce IL-4, but no or limited IFN-~/. We show here that all the allergen-specific TCC isolated from peripheral blood mononuclear cells (PBMNC) of a patient with severe atopic disease produced high amounts of IL-4 in association with IL-3, IL-5 and granulocyte monocyte- colony stimulating factor (GM-CSF), whereas they virtu- ally lacked the ability to produce IFN-7. Materials and methods Reagents Dermatophagoides pteronyssinus group I (Der p I) antigen was kindly provided by G. Mistrello (Lofarma Allergeni, Milan, Italy). Phorbol 12-myristate 13-acetate (PMA) was purchased from Sigma (St. Louis, MO). OKT3 (anti-CD3) monoclonal antibody (mAb) was purchased from Ortho (Raritan, NJ). Recombinant IL-2 was kindly provided by Eurocetus (Milan, Italy). Patient PBMNC were obtained from one 22-year-old man with severe atopic dermatitis and extrinsic asthma. He showed immediate-type hypersensitivity to house dust mite and grass pollen extracts and had high levels of IgE in the serum (20,000 units/ml). Generation of Der p 1-specific TCC Der p I-specific TCC were generated using a technique previously described in detail [6]. Briefly, PBMNC were stimulated with Der p I (25 lag/ml) for 5 days. Subsequently, recombinant IL-2 (20 units/ml) was added and the culture was maintained for an additional 7 days. Viable T blasts were seeded at 0.3 cell per well in 96-well round-bottomed plates in the presence of l0 s irradiated (5,000 rads) allogeneic spleen MNC as feeder cells, 1% (vol/vol) phytohemagglutinin (PHA) and recombinant IL-2 (20 units/ml). Growing microcultures were then expanded at weekly intervals with 105 irradiated feeder cells and IL-2. To propagate established TCC,