0022-1767/86/1369-3204802.00/0 THE JOURNAL OF IMMUNOLOGY Copyrlght 0 1986 by The American Assoclatlon of Immunolcglsts Vol. 136, No. 9, May 1, 1986 Printed Ln U.S.A. PROLIFERATION OF PHENOTYPICALLY IMMATURE HUMAN THYMOCYTES WITH AND WITHOUT INTERLEUKIN 2 RECEPTORS MAURO PIANTELLI,' LUIGI M. LAROCCA, FRANCESCA B. AIELLO, NICOLA MAGGIANO, ARNALDO CARBONE, FRANC0 0. RANELLETTI," AND PIER0 MUSIANI From the Departments of Pathology and 'Histology, Universita' Cattolica, Largo F. Vita 1, 00168 Roma, Italia Previous studies have indicated that the human thymus is composed of several discrete compart- ments. Cortical thymocytes are reactive with the monoclonal antibody anti-T6, whereas most med- ullary cells, unreactive with anti-T6, stain brightly with anti-T3, which defines mature T cell popula- tions. Only a minor thymocyte population lacks both T3 and T6 but expresses T11 antigens. Within the thymus, several proliferating lymphoblasts are present. In addition a distinct subset shows the capacity to proliferate in response to mitogens. By continuous Percoll density gradient centrifugation, we have obtained a cell fraction comprising the vast majority of cells able to proliferate spontaneously or after PHA stimulation. By a panning procedure performed with anti-T3 and anti-T6 antibodies, three phenotypically distinct thymocyte subsets were separated from this fraction, and their func- tional capabilities were tested. The spontaneous proliferating activity was found to be mainly attrib- utable to thymocytes unable to respond to mitogen, expressing the cortical T6 marker and lacking re- ceptors for IL 2. TSpositive cells are able to respond to mitogen. However,these thymocytes are incapa- ble of producing the adequate amount of IL 2 re- quired to fully saturate their intrinsic proliferative capability. Surprisingly, the phenotypically least mature intrathymic T lymphocytes (T3 and T6 neg- ative] respond to phytomitogen, at least in part, in an interleukin-dependent manner. It is noteworthy that a large proportion of these T3- and T6-negative thymocytes express IL 2 receptors and class 11 MHC antigens without in vitro activation. These novel findings have potential implications in the context of current models of differentiation pathways within the human thymus. In man, discrete stages of intrathymic ontogeny have been defined on the basis of monoclonal antibody probes directed at unique T lineage-specific surface antigens (1 - 3). Thus, the earliest identifiable T cell (stage I) expresses the sheep erythrocyte binding glycoprotein, T1 1 , but not T6 or T3 antigens. With further maturation, the T6 mol- Received for publication June 26, 1985. Accepted for publicationJanuary 23, 1986. payment of page charges. This article must therefore be hereby marked The costs of publication of this article were defrayed in part by the advertisement in accordance with 18 U.S.C. Section 1734 solely to indi- cate this fact. "Oncologla." ' This work was supported by a grant from CNR, Progetto Finalizzato reprints should be addressed. 2Recipient of a grant from M.P.I. (40%). and to whom requests for ecule is acquired (stage 11). Both stage I and stage I1 thymocytes are mainly located in the thymic cortex and represent about 75% of the total cell population. The more mature intrathymic pool (stage 111) consists of thy- mocytes that lack the cortical T6 marker and express T3 membrane molecules (a common feature of mature T lymphocytes). Stage 111 cells are mainly found in the thymic medulla. It has been difficult to correlate pheno- typic expression with functional stages of intrathymic cell differentiation because thymocytes are relatively in- active in in vitro assays of T lymphocyte function. Thus human thymocytes respond poorly to mitogens or allo- geneic cells (4-6). Most of our knowledge on the role of the thymus in the ontogeny of T lymphocytes derives from experiments carried out in mice. There is ample evidence that nonlym- phoid components of the gland influence the antigen recognition repertoire of developing pre-T cells. As a con- sequence, the mature T lymphocytes derived from them are able to recognize foreign antigens in the context of self major histocompatibility complex (MHC) products (7, 8). This developmental process seems to require a rapid cell division induced in primitive lymphoblasts by the interaction with the thymic stroma (9, 10). However, the thymic lymphoblast population comprises not only T cell precursors, but also thymocytes expressing cortical or medullary markers (1 1). It was recently reported that proliferating thymic lymphoblasts are among the largest and the lightest cells found in both mouse and human thymus (1 1, 12). Physical separation methods, and in particular centrif- ugation to equilibrium in density gradient, have been shown to reveal a great deal of subpopulation complexity within thymus cells, giving very effective separation of functional from nonfunctional elements (1 2- 17).By con- tinuous Percoll density gradient centrifugation we sepa- rated the lightest cell fraction, which accounted for 10 to 15% of the total thymocyte population and comprised the vast majority of cells able to proliferate spontaneously or after mitogen stimulation. By panning procedures per- formed with monoclonal antibodies, three phenotypically distinct thymocyte subsets were obtained from this frac- tion, and their functional capabilities were tested. The main findingsof this work can be summarized as follows: most of the spontaneous proliferating activity was found tobe mainly attributable to thymocytes lack- ing receptors for interleukin 2 (IL 2). unable to respond to mitogen, and expressing the cortical T6 marker; the phenotypically least-mature intrathymic T lymphocytes (stage I, T3- and T6-negative cells) expressed IL 2 recep- 3204