Study of the distribution pattern of soluble HLA class I heavy chains in CML patients under IFN therapy using SDS-PAGE followed by western blot

Patients treated with IFN showed enhanced soluble HLA class I plasma concentrations [1]. In order to determine the impact of IFN therapy on the distribution pattern of the predominant molecular weight variants of sHLA-1 we studied plasma probes of 13 patients suffering from CML using SDS-PAGE followed by immunoblotting. The patients were treated with INF-alpha (n=5) alone or in combination with IFN-gamma (n=6) or TNF-alpha (n=2). Immunoblotting was performed with mab HC-10, recognizing an epitope on denatured HLA class I heavy chains. In order to determine the part of the soluble HLA class I antigens missing the intracellular fragment an antiserum towards the COOH-terminus of the heavy chain (RaCT) was employed. Using the mab HC-10 all patients showed heavy chains with 43 and 34 kd molecular weight. In 11 out of 13 patients (85%) a 39 kd band and in 8 out of 13 patients (62%) a - some rather weak - 36 kd band could be observed. Only one patient showed a band with 32 kd. Using the antiserum RaCT (only done in 8 patients) all plasma probes showed a 43 kd band but only in 7 out of 8 (88%) a 39 kd heavy chain could be observed. The comparison of the presence and intensity of the 39 kd molecular weight clearly demonstrated different epitopes for these two antibodies. The quantitation of soluble HLA class I plasma levels by means of an ELISA system showed before therapy values with a range of 0.38-5.68 mu g/ml (mean: 1.88 mu g/ml) followed by a decrease of the concentrations during 2-4 hr after beginning of treatment. After that the plasma levels increased reaching a maximum within the next 6 weeks. The changes of the plasma concentrations mostly are in line with band intensities after immunoblotting with mab HC-10. These results clearly demonstrated variations in plasma concentrations but no changes in individual distribution pattern of the sHLA-I heavy chains under IFN therapy.