Transplant. Rev. (1915), Val. 25 Published by Munksgaard, Copenhagen, Denmark No part may be reproduced by any process without written permission from the author(s)
Lymphocyte Subpopulations in the Thymus W. D R O E G E & R. ZUCKER
1. Introduction Practically all vertebrates have a thymus. This organ provides the inductive stimulus for stem cells to mature into immunocompetent cells. After maturation in the thymus, these cells are exported into the peripheral lymphatic system and constitute the pool of 'thymus derived lymphocytes' (Miller & MitcheU 1969). The thymus derived lymphocytes were found to carry a variety of immunological functions, such as helper activity for antibody responses (Miller & Mitchell 1969), MLC reactivity (Takiguchi et al. 1971b), reactivity in cell mediated in vivo and in vitro response (Miller 1966, Cooper et al. 1966, Wagner et al. 1972), and suppressive activity in various types of immune responses (for review see Droege 1973a, Gershon 1974, Playfair 1974). Experimental evidence is now accumulating that different functions are carried by different sublines of T-cells, and that different sublines might be detected not cmly in the periphery but possibly already in the thymus itself. This is a brief review about the experimental data that led to the distinction of lymphocyte subpopulations in the thymus. The possible existence of different cell lineages in the thymus will be discussed. 2. The Lymphocyte Pools of the Cortex and the Medulla The lymphocyte compartments of the thymus are schematically illustrated in Table I. The thymus of most species contains two distinct regions: the cortex and the medulla. In young mice, the cortex shares about 86 % of Basel Institute for Immunology, Basel, Switzerland and Papanicolaou Cancer Research Institute, Miami, Florida, U.SA.
W, DROEGE & R. ZUCKER TABLE I Schematic illustration of the lymphocyte •compartments in the thymus
Medium sized lymphocytes (about 10 %) Small lymphocytes (about 87 %)
Medullary lymphocytes (about 15 %)
Stem cell?
Stem cells?
Cell type IV: progenitor cells fo:- small thymic lymphocytes
Early cortical small cell {cell type JI in mice, cell type I in chickens) may contain suppressive activity
Late cortical small cell (cell type I in mice, cell type Ii in chickens) responsible for liite T cell functiiMis?
Cell type III (hydrocortisone resistant, light buoyant density) carries a number of biological activities
Developmental pathways
Large lymphocytes (about 3 %)
Cortical lymphocytes (about 85 %)
the thymic volume and the medulla about 14 % (Bryant 1972). A similar high ratio for cortex versus medulla is fotnd in the young human thymus (Simpson et al. 1975) and in the young chicken thymus (Warner 1964). About 87 % of the total thymic lymphocytes in the mouse are small lymphocytes with a nuclear diameter of ;5-7 fim in conventional smears (Bryant 1972). The medium and large lymphocytes (nuclear diameter in conventional smears 8-12 fxia) comprise about 13 % of the total lymphocyte pool of the mouse thymus (Bryant 1972). The larger cells are mitotically active and feed into the pool of small lymphocytes, the majority of which are non prohferating (Bryant 1972, Potmesil & Goldfeder 1973). Whether the cortical and the medullary lymphcxryte pools are independent in respect to these developmental pathways, or whether one is feeding into the other is not clear. The fact is that both pools contain medium and large lymphocytes (the medulla somewhat more (26 %) than the cortex (11 %), and that the larger cells in both regions are mitotically active (PotmesU & Goldfeder 1973). Essentially similar figures where obtained by Shortman & Jackson (1974). A cell fraction which is relatively insensitive to cytotoxic anti-Thy 1.2 antisenmi and which is believed to correspond to the medullary lymphocyte pool is found to contain about twice as many large cells as the highly sensitive (cortical) pool. Large cells in both pools are mitotically active:
LYMPHOCYTE SUBPOPULATIONS IN THE THYMUS
5
They incorporate tritialed thymidine extremely fast and feed into the pools of small lymphocytes, the anti-Thy 1.2-sensitive and the relatively insensitive pools being labelled at the same rate and without lag phase. The data suggest independent developmental pathways from large to small cells both in the cortical and the medullary compartments (Shortman & Jackson 1974). There is no satisfactory method for the preparative separation of cortical and medullary lymphocytes. Hydrocortisone treatment in mice eliminates most of the cortical cells (Ishidate & Metcalf 1963), but seems also to eliminate all larger cells including those from the medulla (see chapter 5, Figures 7 and 8). Single injections of hydrocortisone in chickens, on the other hand, do not completely eliminate all cortical cells (Zucker et al. 1973). The normal adult chicken thymus, however, contains practically no cortex and yields good preparations of medullary lymphocytes (Warner 1964, Droege et al. 1974a). Many studies in mice take advantage of the differential sensitivity to specific antisera like anti-H2, anti-TL or anti-Thy 1.2 (Aoki et aL 1969, Leckband 1970, Leckband & Boyse 1971, Smith 1972, Stobo 1972, Levey & Burleson 1972, Shortman et al. 1972, Shortman & Jackson 1974). The best discrimination apparently is obtained with the controlled anti-Thy 1.2-cytotoxic treatment (Shortman & Jackson 1974). The differential staining with fluorescent anti-Thy 1.2 serum seems to correlate less well with the cortical and medullary pool: The large cells from the medulla apparently stain bright and thus are indistinguishable from the cortical lymphocytes (Fathman et al. 1975). 3. The Cortical Small Lymphocytes ('Early Small Cortical' and 'Late Small Cortical' Lymphocytes) Roughly three quarters of all lymphocytes in the fully developed thymus of the young mouse or the young chicken are cortical small lymphocytes. The heterogeneity of the small thymocytes of mice and chickens has been studied by using size distribution analysis in combination with preparative cell electrophoresis and bovine serum albumin (BSA) density gradient centrifugation (Droege et al. 1974a, b, Zucker et al. 1973). In both species, it was possible to distinguish three major subpopulations of small lymphocytes. The two-dimensional distribution pattems ('fingerprints') in Figure 1 (left side) and 2 (left side) illustrate the three cell types from the young mouse thymus. The cells in the low electrophoretic mobility region (relative electrophoretic mobility 0.9-1.0) and in the high density fractions (1.070-1.075 g/cm^) show a constant modal size which is defined as rel. electronic volume 1.0 (electronic volume = volume as given by the size distribution analyzer). This cell population is referred to as cell type I.
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Lymphocyte Subpopulations in the Thymus W. D R O E G E & R. ZUCKER
1. Introduction Practically all vertebrates have a thymus. This organ provides the inductive stimulus for stem cells to mature into immunocompetent cells. After maturation in the thymus, these cells are exported into the peripheral lymphatic system and constitute the pool of 'thymus derived lymphocytes' (Miller & MitcheU 1969). The thymus derived lymphocytes were found to carry a variety of immunological functions, such as helper activity for antibody responses (Miller & Mitchell 1969), MLC reactivity (Takiguchi et al. 1971b), reactivity in cell mediated in vivo and in vitro response (Miller 1966, Cooper et al. 1966, Wagner et al. 1972), and suppressive activity in various types of immune responses (for review see Droege 1973a, Gershon 1974, Playfair 1974). Experimental evidence is now accumulating that different functions are carried by different sublines of T-cells, and that different sublines might be detected not cmly in the periphery but possibly already in the thymus itself. This is a brief review about the experimental data that led to the distinction of lymphocyte subpopulations in the thymus. The possible existence of different cell lineages in the thymus will be discussed. 2. The Lymphocyte Pools of the Cortex and the Medulla The lymphocyte compartments of the thymus are schematically illustrated in Table I. The thymus of most species contains two distinct regions: the cortex and the medulla. In young mice, the cortex shares about 86 % of Basel Institute for Immunology, Basel, Switzerland and Papanicolaou Cancer Research Institute, Miami, Florida, U.SA.
W, DROEGE & R. ZUCKER TABLE I Schematic illustration of the lymphocyte •compartments in the thymus
Medium sized lymphocytes (about 10 %) Small lymphocytes (about 87 %)
Medullary lymphocytes (about 15 %)
Stem cell?
Stem cells?
Cell type IV: progenitor cells fo:- small thymic lymphocytes
Early cortical small cell {cell type JI in mice, cell type I in chickens) may contain suppressive activity
Late cortical small cell (cell type I in mice, cell type Ii in chickens) responsible for liite T cell functiiMis?
Cell type III (hydrocortisone resistant, light buoyant density) carries a number of biological activities
Developmental pathways
Large lymphocytes (about 3 %)
Cortical lymphocytes (about 85 %)
the thymic volume and the medulla about 14 % (Bryant 1972). A similar high ratio for cortex versus medulla is fotnd in the young human thymus (Simpson et al. 1975) and in the young chicken thymus (Warner 1964). About 87 % of the total thymic lymphocytes in the mouse are small lymphocytes with a nuclear diameter of ;5-7 fim in conventional smears (Bryant 1972). The medium and large lymphocytes (nuclear diameter in conventional smears 8-12 fxia) comprise about 13 % of the total lymphocyte pool of the mouse thymus (Bryant 1972). The larger cells are mitotically active and feed into the pool of small lymphocytes, the majority of which are non prohferating (Bryant 1972, Potmesil & Goldfeder 1973). Whether the cortical and the medullary lymphcxryte pools are independent in respect to these developmental pathways, or whether one is feeding into the other is not clear. The fact is that both pools contain medium and large lymphocytes (the medulla somewhat more (26 %) than the cortex (11 %), and that the larger cells in both regions are mitotically active (PotmesU & Goldfeder 1973). Essentially similar figures where obtained by Shortman & Jackson (1974). A cell fraction which is relatively insensitive to cytotoxic anti-Thy 1.2 antisenmi and which is believed to correspond to the medullary lymphocyte pool is found to contain about twice as many large cells as the highly sensitive (cortical) pool. Large cells in both pools are mitotically active:
LYMPHOCYTE SUBPOPULATIONS IN THE THYMUS
5
They incorporate tritialed thymidine extremely fast and feed into the pools of small lymphocytes, the anti-Thy 1.2-sensitive and the relatively insensitive pools being labelled at the same rate and without lag phase. The data suggest independent developmental pathways from large to small cells both in the cortical and the medullary compartments (Shortman & Jackson 1974). There is no satisfactory method for the preparative separation of cortical and medullary lymphocytes. Hydrocortisone treatment in mice eliminates most of the cortical cells (Ishidate & Metcalf 1963), but seems also to eliminate all larger cells including those from the medulla (see chapter 5, Figures 7 and 8). Single injections of hydrocortisone in chickens, on the other hand, do not completely eliminate all cortical cells (Zucker et al. 1973). The normal adult chicken thymus, however, contains practically no cortex and yields good preparations of medullary lymphocytes (Warner 1964, Droege et al. 1974a). Many studies in mice take advantage of the differential sensitivity to specific antisera like anti-H2, anti-TL or anti-Thy 1.2 (Aoki et aL 1969, Leckband 1970, Leckband & Boyse 1971, Smith 1972, Stobo 1972, Levey & Burleson 1972, Shortman et al. 1972, Shortman & Jackson 1974). The best discrimination apparently is obtained with the controlled anti-Thy 1.2-cytotoxic treatment (Shortman & Jackson 1974). The differential staining with fluorescent anti-Thy 1.2 serum seems to correlate less well with the cortical and medullary pool: The large cells from the medulla apparently stain bright and thus are indistinguishable from the cortical lymphocytes (Fathman et al. 1975). 3. The Cortical Small Lymphocytes ('Early Small Cortical' and 'Late Small Cortical' Lymphocytes) Roughly three quarters of all lymphocytes in the fully developed thymus of the young mouse or the young chicken are cortical small lymphocytes. The heterogeneity of the small thymocytes of mice and chickens has been studied by using size distribution analysis in combination with preparative cell electrophoresis and bovine serum albumin (BSA) density gradient centrifugation (Droege et al. 1974a, b, Zucker et al. 1973). In both species, it was possible to distinguish three major subpopulations of small lymphocytes. The two-dimensional distribution pattems ('fingerprints') in Figure 1 (left side) and 2 (left side) illustrate the three cell types from the young mouse thymus. The cells in the low electrophoretic mobility region (relative electrophoretic mobility 0.9-1.0) and in the high density fractions (1.070-1.075 g/cm^) show a constant modal size which is defined as rel. electronic volume 1.0 (electronic volume = volume as given by the size distribution analyzer). This cell population is referred to as cell type I.
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