Volume 108, number I FEBS LETTERS December 1979 zyxwvutsr THY-l HETEROGENEITY OF MOUSE THYMOCYTES S. CARLSSON, T. STIGBRAND and B. WINBLAD* zyxwvutsrqponmlkjihgfedcbaZYX Departmerlts of Physiological Chemistry and *Pathology, University, o,f Umeii, S-901 87 Umeii, Sweden Received 5 October 1979 1. Introduction The Thy-l molecule (formerly called theta) is the major membrane antigen of mouse and rat thymocytes [ 1,2]. Large amounts are also found in brain tissue of these species and to a lesser extent on bone marrow cells of the rat, but not of the mouse [3]. On the other hand, Thy-l antigen is expressed on peripheral T-lymphocytes only of the mouse [4] and is widely used for serological differentiation of T- and B-lym- phocytes of the mouse. Following neonatal thymectomy a dramatic decrease of the proportion of Thy-l positive peripheral lymphocytes is observed together with a decrease in cell-mediated immunity [5]. Furthermore physiological maturation of potential T-lymphocytes by passage through the thymus, a process which might depend on soluble thymic fac- tors, can be followed by appearance of Thy-l antigen [6]. Despite the obvious importance of this molecule, little is known of the chemical nature of mouse Thy-l which is still a matter of controversy [7]. In the rat, Thy-l has been described as a glyco- protein with mol. wt 18 000 [8], and it seems likely that the corresponding molecule of the mouse is of similar nature [7]. The function of Thy-l is still unclear but a possible role in cell-cell interactions, involving the carbohydrate part, has been discussed. Antisera with specificity for Thy-l antigen do not necessarily detect differences in carbohydrate constitutents of glycoproteins. By use of lectins, with defined specific- ity for different sugar derivatives, heterogeneity in the carbohydrate part of Thy-l from rat has been found [9]. Mouse Thy-l has not, so far, been shown to reveal such diversity. The possible existence of Thy-l heterogeneity of mouse thymocytes is challenging 116 and might have functional implications. This paper describes such a heterogeneity. 2. Materials and methods 2 .l . Cell and Thy - I preparation Thymus glands from 2-4 month old C57BL mice (expressing Thy-l .2 antigen) were used as the source of thymocytes. Cells were washed twice in Hank’s medium and lysed in 0.1 M Tris-HCl buffer (pH 8.0) containing 0.5% Na-deoxycholate. This concentration of detergent has been found not to interfere with the assay for quantitative determination of Thy-l [lo]. The sample (corresponding to -40 X 1O6 cells/ ml) was sonicated twice for 60 s at +4”C and left for 1 h at +4”C with slight agitation. After centrifugation 50 min at 100 000 X g the supernatant was collected. 2.2.Antiserum The antiserum was raised in rabbits against mouse brain homogenate [ 1 I] and was shown to be specific for Thy-l after appropriate absorption [lo]. 2.3. Thy-1 assay Thy-l was quantitated as in [lo]. Briefly, the material to be assayed was diluted in ‘log dilutions in Hanks’ medium complemented with 10% bovine serum albumin. After addition of antiserum the mixture was incubated at +4”C for 1 h. The amount of anti-Thy-l antibodies consumed was measured by adding guinea pig serum as source of complement and 51Cr-labelled thymocytes. The mixture was incubated for 1 h at 37°C. The antigen dilution giving 50% lysis was used as a measure of antigen activity. ElsevierfNorth-Holland Biomedical Press