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The phenotype of the lymphoid cell component of 35 thymomas was investigated by analyzing cryostat sections and lymphocyte suspensions. The morphology in each case was determined by examining multiple tissue samples from different parts of the tumor. Structural heterogeneity was shown in 14 thymomas, and a homogeneous morphology of cortical or medullary or mixed types in the others. To assess whether this heterogeneity was correlated with differences in the lymphoid phenotype, we analyzed both lymphocyte suspensions and frozen sections from the same samples. Phenotypical differences in the suspensions of each thymoma in the heterogeneous group were noted and similar differences were also observed in the cryostat sections. Phenotypical abnormalities were found in some thymomas. They consisted of the simultaneous expression of cortical and medullary markers, which was most marked in the heterogenous mixed-type thymomas invading the lung. Furthermore, the global phenotype was tested on a pool of lymphocyte suspensions in all thymomas. This procedure distinguished cortical, medullary and intermediate cortico-medullary immunophenotype models which closely correlated with the tumor histology. It was concluded that, due to the frequent structural and immunological heterogeneity of thymomas, correct assessment of their lymphoid component requires a two-step analysis. This comprises: 1) individual suspensions from samples taken from different areas of the same thymoma, and 2) a pool of these suspensions. The first step will reveal the different immunological characteristics. In the second, the lymphocyte phenotype, which may vary widely throughout the tumor, will be represented in its totality. These findings may be of great help in predicting clinical patterns, especially possible malignant evolution.

The primary role of the T-cell system in immune-complex glomerulonephritis induced by intravenous weekly injections of human serum albumin (HSA) in rats has been demonstrated. The development of histological, ultrastructural and immunological glomerular alterations which are clearly recognizable in intact animals was prevented by neonatal thymectomy. In vitro tests of cellular immunity (LIF and PHA responsiveness) revealed a close relationship between the involvement of functioning T-cell subpopulations (at least T-helper) and the development of the classic glomerulonephritic pattern. In other words HSA antigen recognition by T lymphocytes, their cooperation with B lymphocytes, and the activation of the latter with related antibody response represent the immunological sequence which leads to the formation of the soluble circulating immune-complexes responsible for the glomerular injury. Our findings suggest that the same immunological sequence can represent the pathogenetic basis for many forms of glomerulonephritis in which T-dependent antigen stimulation is demonstrable. Our data are also discussed in the light of results obtained by others in immuneglomerulonephritis induced in nude athymic mice.

The extent and distribution of alfa-naphthyl acetate esterase (ANAE) activity in both normal thymuses and thymuses or thymomas from patients with myasthenia gravis were investigated by enzyme histochemistry. In all a closely similar distribution of ANAE activity was recognizable. ANAE positive thymic lymphocytes were distributed preferentially at the cortico-medullary junction and in the medulla. Their numbers at these sites were higher in myasthenic patients than in normal adults (P less than 0.001) and lowest in fetuses. The relative numbers of cells showing both ANAE activity and EN-rosette-forming capacity was lower in thymocytes than peripheral lymphocytes but was increasing higher in thymuses from fetal, adult and myasthenic patients and in thymomas. These findings are discussed in relation to the stage of maturity of thymocytes and the importance of intrathymic immunological challenge in the attainment of these cells of functional properties usually acquired in the periphery.

Tissue sections of lymph nodes, appendices and tonsils, together with smears of immunologically separated peripheral lymphoid cells from a B-CLL and lymphomatous cells from an immunocytoma were submitted to combined enzyme cytochemical investigations with acid alpha-naphthyl acetate esterase (ANAE), beta-glucuronidase (B-G), acid phosphatase (AcPh), adenosine triphosphatase (ATPase), a,d 5'nucleotidase (5'N). T-cells were Acph+, ATPase- and 5'N-. The vast majority of T- and B-cells displayed ANAE and B-G activities with two distinct staining patterns (T-like and B-like pattern). A high proportion of lymphoid cells in the germinal centre (G.C.) and the vast majority of lymphoid cells in the mantle-zone (M.Z.) were shown to belong to B-cell system because of the expression of ATPase and 5'N in their membranes. Some lymphoid cells positive for ANAE and B-G with a B-like pattern and for AcPh were recognizable in the G.C. In the M.Z. only a few lymphoid cells being ANAE+, with a T-like pattern, and AcPh+ were shown to belong to the T-cell system. In contrast, in this zone a high proportion of small lymphoid cells (64% +/- 10%) showed ANAE activity, mostly with B-like pattern. Therefore, these findings indicate that in the M.Z. a high proportion of B-cells ATPase+ and 5'N+ also display ANAE activity. By comparison of the results obtained from lymphoid tissue sections, B-CLL and immunocytoma cell suspensions and normal circulating lymphocytes we can conclude that B-ANAE-positive cells of the M.Z. do not usually appear in the peripheral blood. They circulate in large numbers only in some pathological conditions (like our reported B-CLL). Therefore, B-ANAE-positive lymphoid cells of the mantle, with a B-like staining pattern, include a wide range of subsets which exclude large lymphoid cells and plasma cells.

The lymphoid tissue provides the most productive conditions to the application of immunohistochemical techniques. This tissue is composed of many B and T phenotypically different cell populations ranging from stem cells to completely differentiated lymphocytes which can be identified by using monoclonal antibodies. These techniques also are of great help in classifying the malignant lymphocyte counterparts and, thus, in defining diagnostic and prognostic characters of different lymphomas/leukaemias.

Twenty-six human thymomas were studied in an attempt to correlate their morphological appearance with the type and degree of T-lymphocyte maturation, as determined by acid alpha-naphthyl-acetate esterase (ANAE) activity and immunological analysis. Four normal human thymuses were used for purposes of comparison. Two morphological patterns were identified in the thymomas. The distinction was based largely on similarities between the neoplastic epithelial cells and normal cortical and medullary epithelial cells, and on the relative proportions of epithelial cells and lymphocytes. By these criteria "medullary" and "cortical" patterns were identified. In several thymomas both patterns were present in the same tumor ("mixed-type pattern"), producing alternating dark cortical-like areas and lighter foci of medullary differentiation. A good correlation was found between the two patterns and the phenotype of the T-associated lymphoid component. ANAE activity, which was completely lacking in normal cortical thymocytes, was almost absent in the phenotypically immature T-cells of cortical-type thymomas. By contrast, in the medullary-type thymomas, T-cells showed immunological features in common with medullary thymocytes. This was characterized by strong ANAE activity in the majority of cells with a staining pattern corresponding to that of peripheral T-lymphocytes. In addition, most of the proliferating epithelial cells in medullary-type thymomas stained strongly with anti-cytokeratin and anti-epidermal-type keratin antisera. In the mixed-type thymomas the epithelial cell morphology and the immunohistochemical and enzymic features of the T-cells were found to be closely related to the respective cortical--or medullary-like areas. It was concluded that the various characteristics of normal thymic cortex and medulla studied are also present in thymomas. In particular, in medullar-type thymomas the presence of many of the features of normal thymic medulla, such as a squamous cell component, macrophages and interdigitating reticulum cells, may constitute a microenvironment which operates actively in T-cell education. This may account for the functional activities, characteristic of peripheral T-lymphocytes, which T-lymphocytes attain in these thymomas.