Cutting Edge: A possible role for CD4+ thymic macrophages as professional scavengers of apoptotic thymocytes

A vast majority of thymocytes are eliminated during T cell development by apoptosis. However, apoptotic thymocytes are not usually found in the thymus, indicating that apoptotic thymocytes must be eliminated rapidly by scavengers. Although macrophages and dendritic cells are believed to play such role, little is known about scavengers in the thymus. We found that CD4(+)/CD11b(+)/CD11c(-) cells were present in the thymus and that they expressed costimulatory molecules for T cell selection and possessed Ag-presenting activity. Moreover, these CD4(+)/CD11b(+) cells phagocytosed apoptotic thymocytes much more efficiently than thymic CD4(-)/CD11b(+) cells as well as activated peritoneal macrophages. CD4(+)/CD11b(+) cells became larger along with thymus development, while no such change was observed in CD4(-)/CD11b(+) cells. Finally, engulfed nuclei were frequently found in CD4(+)/CD11b(+) cells. These results strongly suggest that thymic CD4(+)/CD11b(+) cells are major scavengers of apoptotic thymocytes.     

Avian thymic accessory cells

On the basis of morphologic criteria and ingestion of latex particles, two basic types of accessory cells can be identified from quail and chick thymuses, dendritic cells, and macrophages. By using embryonic grafting techniques, we show that cells of this lineage enter the thymus during the initial colonization of the epithelial thymic rudiment by hemopoietic cells, and within a few days differentiate into cells exhibiting properties of glass adherence, Ia expression, and formation of rosettes with thymocytes. It appears that the precursors of this lineage undergo extensive, but finite, proliferation and are eventually replaced by further influx of the accessory cell lineage. In chimeric grafts, quail thymocytes were seen forming rosettes with chick accessory cells, and vice versa, indicating, as in the interaction between the epithelial cells and thymocytes, that the molecules involved in thymocyte-accessory cell association can interact across species barriers in our system.     

Development of CD4+ macrophages from intrathymic T cell progenitors is induced by thymic epithelial cells

It was recently demonstrated that there are CD4(+) macrophages, which exhibit strong phagocytic activity, in the thymus. They are suggested to play an important role for the elimination of apoptotic thymocytes. However, the origin and nature of CD4(+) macrophages in the thymus remain unexplored. In this study, we describe that the most immature intrathymic progenitors (CD25(-)/CD44(+)/FcR(+)) give rise to CD4(+) macrophages by oncostatin M-responsive thymic epithelial cells (ORTEC) in an IL-7-dependent manner. Neither conditioned medium of ORTEC nor a mixture of cytokines induced CD4(+) macrophages, and oncostatin M receptor was not expressed in thymocytes, suggesting that the development of CD4(+) macrophages from the immature thymocytes requires a direct interaction with ORTEC. These results collectively suggest that the development of CD4(+) macrophages from the intrathymic T cell progenitors is induced by thymic epithelial cells.     

The identification of thymic nurse cells in vivo and the role of cytoskeletal proteins in thymocyte internalization

Much debate has been generated about the existence of thymic nurse cells within the thymus. Until now, the authenticity of an epithelial cell capable of internalizing developing thymocytes within the thymic cortex has been in question. Here, we use the thymic nurse cell-specific monoclonal antibody, ph91, to define the in vivo location of thymic nurse cells. For the first time, thymic nurse cells enclosing several thymocytes were detected in the subcapsular region of the thymic cortex in a “honeycomb-like” configuration. In vitro studies show the internalization process using digitalized time-lapse microscopy. Internalized thymocytes have also been reported to interact with macrophages within the TNC complex. The cytoplasmic interaction between thymocytes and macrophages was detected using time-lapse microscopy. Using fluorescence microscopy, we show polymerization of actin within macrophages at the contact point with thymocytes, which is indicative of an immunological synapse. Microfilaments and microtubules within TNCs were shown to be associated with thymocyte binding and internalization, but neither interacted with macrophages. Also, we provide data to show that thymocytes are actively involved in the internalization process. These experiments show for the first time the existence of thymic nurse cells within the thymic microenvironment. They provide a visual documentation of thymocyte uptake by thymic nurse cells, and define an interaction between thymocytes and macrophages within the TNC complex.     

The professional phagocytes of sea bass (Dicentrarchus labrax L.): cytochemical characterisation of neutrophils and macrophages in the normal and inflamed peritoneal cavity

In order to identify the phagocytic cells of sea bass, the peritoneal leucocyte population of fish injected intraperitoneally with Photobacterium damselae subspecies piscicida was studied by light microscopy using cytocentrifuge preparations stained by the Antonow technique for peroxidase detection. Among the leucocytes present in the peritoneal exudate of the infected fish (macrophages, neutrophils, eosinophilic granular cells, lymphocytes and thrombocytes), macrophages and neutrophils were the only phagocytic cells. Neutrophils were easily distinguished from macrophages in Antonow stained preparations by the pattern of peroxidase positivity. Using ultrastructural cytochemistry, neutrophils were found to have abundant cytoplasmic granules positive for peroxidase and arylsulphatase and were negative for alpha-naphthyl butyrate (ANB) esterase. In contrast, ANB esterase activity was detected in macrophages. These leucocytes were typically negative for peroxidase, but ocasionally, some macrophages with peroxidase or arylsulphatase-positive vacuoles were observed. Both phagocytes had cytoplasmic granules positive for acid phosphatase. Glycogen particles were found in the cytoplasm of the two phagocytic cells, but they were much more abundant in neutrophils. Macrophages were much more abundant than neutrophils in the peritoneal cavity of non-injected sea bass but early after the intraperitoneal injection of bacteria, the number of neutrophils increased quickly and extensively. Higher numbers of intraperitoneally injected bacteria were found inside macrophages as compared to neutrophils because macrophages strongly predominated in the peritoneal population at the time of injection. However, when the bacteria were injected into peritoneal cavities with high numbers of neutrophils (attracted by a previous injection of 12% casein), the percentage of neutrophils with phagocytosed bacteria increased, approaching that of infected macrophages. Taken together, these results show that in sea bass, as in many other organisms, in addition to macrophages, neutrophils are important phagocytic cells, the relative participation of each of the two phagocytes in defense mechanisms against infection depending on the opportunity to encounter the invading infectious agents.     

Circulating macrophages as well as developing thymocytes are enclosed within thymic nurse cells.

Both thymic nurse cells (TNCs) and macrophages have been reported to function as antigen-presenting cells during the process of MHC restriction. Negative selection, which results in the apoptosis of potentially autoreactive thymocytes, is believed to be associated with both macrophages and TNCs in the cortex. Both cell types have also been reported to ingest thymocytes undergoing positive and negative selection. However, macrophages ingest apoptotic thymocytes, while TNCs have been shown to internalize viable cells. A subset of the TNC-engulfed population is allowed to mature and is released, while the remaining fraction becomes apoptotic and is absorbed within the TNC cytoplasm through lysosomal activity. A recent report described a subset of rat TNCs that contain macrophages as well as thymocytes within their cytoplasm. We examined freshly isolated TNCs from C57BL/6 mice and found that, of the TNC population recovered, 1.7% contained macrophages within its cytoplasm. There also were macrophages tightly bound but not internalized into the multicellular structure at a rate of 2.9%. The total association of macrophages with TNCs was approximately 4.6%. This unique association of macrophages with TNCs was also observed in vitro when freshly isolated thymocytes (containing macrophages) were added to cultures of cells from the TNC cell line tsTNC-1. The macrophage-TNC interaction was found to be dynamic, with macrophages moving rapidly into and out of TNCs containing cytoplasmic thymocytes. Macrophages within TNCs showed a close association with cytoplasmic thymocytes. We then labeled peritoneal macrophages with CFDA SE, a cell tracking dye, and returned them to the mouse peritoneum. Within 1 h, labeled macrophages were detectable in the thymus. This is the first investigation to show a direct interaction between peripheral macrophages and TNCs. These results suggest that TNCs and macrophages work together as antigen-presenting cells.     

Development and characterization of a continuous macrophage cell line,LRTM, derived from thymus of Labeo rohita (Hamilton 1822)

A long-term thymic macrophage cell line from the thymus explants of Labeo rohita designated as LRTM (L. rohita thymic macrophages) was established, which has been maintained in culture for more than 1 yr. This cell line designated LRTM cells have been subcultured for 70 passages. The cells shape was initially long and elongated; with subsequent passages, the cells became short and epithelial like. The cells exhibited optimum growth in L-15 containing 10% fetal bovine serum and also in Dulbecco’s modified Eagle’s medium at 37°C with 5% CO₂ and showed 85+?% viability after 12 mo storage in liquid nitrogen. In addition, cells showed nonspecific esterase and surface expression of Fc receptors for immunoglobulin G and classes I and II major histocompatibility complex antigens. These observations confirmed that this cell line had the morphologic and functional features as a macrophage. The cells exhibited phagocytic activity by engulfing yeast cells as well as fluorescent latex beads, which was demonstrated by scanning electron microscopy and Giemsa staining. The long-term cultured cells show rapid production of reactive oxygen and nitrogen intermediates following stimulation with lipopolysaccharides and phorbol miristate acetate (PMA). Mostly, all the cells were alpha napthyl esterase acetate positive. After stimulation with PMA and lipopolysaccharide, cultured fish macrophages produced reactive oxygen and nitrogen intermediates. The karyotype analysis showed that these cells have a tetraploid karyotype with 100 chromosomes in each cell, indicating that they are normal L. rohita cells. Amplification, sequencing, and alignment of fragments of two mitochondrial genes 12S rRNA from rohu confirmed that the cell line originated from L. rohita. This cell line should be useful for studying the role of thymic macrophages in differentiation and maturation of thymocytes and can be source of macrophage-specific enzymes and cytokines. The macrophage cell line will be invaluable in studies of pathogen/macrophage interactions, the mechanisms of macrophage antimicrobial effector functions and the contribution of macrophages to the specific immune responses of teleosts.     

APPEARANCE AND DISTRIBUTION OF FERRITIN IN MOUSE PERITONEAL MACROPHAGES IN VITRO AFTER UPTAKE OF HETEROLOGOUS ERYTHROCYTES

Mouse peritoneal macrophages have been studied in vitro after ingestion of treated rat, rabbit, or sheep erythrocytes. Under light microscopy, phagocytic vacuoles persist up to 24 h. Macrophages lose benzidine reactivity about 5 h after red cell ingestion, and they become prussian blue positive at 2 days. Ultrastructural studies show little or no ferritin in control macrophages not fed erythrocytes. In contrast, after red cell ingestion, ferritin is widely distributed in the cytoplasmic matrix and in some cytoplasmic granules by 48 h. The Golgi complex, pinocytic vacuoles, endoplasmic reticulum, nuclei, and mitochondria do not contain ferritin. Between 2 and 4 days, ferritin in cytoplasmic granules increases, concomitant with decrease in the ferritin in the cytoplasmic matrix. Evidence is presented suggesting that ferritin in the cytoplasmic matrix is translocated into cytoplasmic granules by autophagy. Polyacrylamide gel studies on macrophages after uptake of red blood cells labeled with radioiron confirm that macrophages produce radiolabeled ferritin by 4 days.     

Thymic microenvironment and cultures derived from mouse thymic explants

Summary Cultures derived from thymus fragments of embryonic (18–19 day old), newborn or one month old C57 BL mice have been characterized functionally (phagocytic and nonspecific esterase activities) and morphologically by means of light, scanning (SEM) and transmission (TEM) electron microscopy. The observations show the heterogeneity of the cell populations composing the monolayers. After a few days incubation macrophages appear as the predominating cell type, while epithelial cells usually constitute no more than 30% of the cells. Experiments designed to determine the fate of lymphocytes adhering to the monolayers lead us to believe (on the basis of SEM morphometric analysis) that the survival of lymphocytes attached either to thymic macrophages or to epithelial cells is improved during the first days of coculture. This survival enhancement does not, however, appear to be a specific inductive effect since a similar survival increase is found when lymphocytes adhere to non-thymic cells. In contrast with the monolayer, the expiant provides a three-dimensional culture system able to preserve intact thymic microenvironmental conditions since numerous lymphocytes are found even in five week old cultures which were not overlaid with thymocytes or spleen cells.     

Effect of cytokines on thymic hematopoietic precursors

Summary We have previously shown that the interaction of thymocytes with thymic accessory cells (macrophages and/or interdigitating cells) is one of the factors required for thymocyte activation. Precursors of both thymic accessory cell and thymocytes are included in the CD4^- CD8^- Mac-1^- Ia^- subpopulation, and their respective maturation and/or activation may be modulated by granulocyte-macrophage colony-stimulating factor, interleukin 1 and interleukin 2. When CD4^- CD8^- thymic cells are activated with granulocyte-macrophage colony-stimulating factor plus interleukin 2, both macrophages and interdigitating-like cells are present, as shown by electron microscopy. When activated with interleukin 1 plus interleukin 2, the interdigitating-like cells is the only accessory cell present. In both culture conditions, large clusters are formed between interdigitating cells and lymphoid cells. These results have led us to propose two-step signals for thymocyte proliferation: first, the maturation of macrophages under granulocyte-macrophage colony-stimulating factor control and the production of interleukin 1, and secondly, the maturation of interdigitating cells under interleukin 1 control, their clustering with thymocytes which are then activated.Abbreviations CFU-S colony-forming units in the spleen - CSF colony-stimulating factor - DC dendritic cells - DN double negative cells (CD4^- CD8^-) - EC epithelial cells - GM-CFC granulocyte/macrophage colony-forming cells - GM-CSF granulocytemacrophage CSF - IDC interdigitating cell - IL-1 interleukin 1 - IL-2 interleukin 2 - MØ macrophage - P-TR phagocytic cell of the thymic reticulum     

Thymic accessory cell complexes in vitro and in vivo: morphological study

Summary Murine thymic macrophages and interdigitating cells, also called thymic accessory cells, were characterized by means of light- and electron microscopy. The cells were studied in suspension, during isolation by enzymatic digestion and in vivo. They were observed as isolated cells or as components of multicellular complexes, some of which were rosettes and were composed of lymphoid cells centered on each type of accessory cell. We also noted other cell complexes including macrophages that resembled classical epithelial nurse cells. We consider that multicellular complexes represent lymphostromal associations already existing in vivo, because we observed them at the periphery of thymic pieces undergoing enzymatic treatment. The heterogeneity of macrophages that we observed in vitro was also noted in vivo. In vivo macrophages were of three types: classical phagocytic cells distributed throughout the gland, cortical elongated cells in close contact with lymphoid blast cells, and atypical nurse cells containing mitotic cells and located in the inner cortex. The morphological aspects of the latter two cell types suggest that cortical macrophages in vivo have other roles: they can be interpreted as images of positive or negative cell selection. We also believe that rosettes are formed by elongated cortical macrophages when they are enzymatically isolated from the thymus.Part of this work was presented at the Second Thymus Workshop, Rolduc, The Netherlands, April 1989     

Characterization of thymic cell subpopulations involved in IL-1- or GM-CSF-induced IL-6 production.

IL-6 has been demonstrated by in vitro studies to be a cytokine involved in thymocyte activation We show herein that thymocytes cultured at high concentrations in the absence of comitogen respond to IL-1 and, to a lesser degree, to GM-CSF, by producing IL-6. This phenomenon disappears rapidly with decreasing cell densities, suggesting the involvement of a minor cellular component of the thymus which may be solely responsible for or cooperate in IL-6 production. We have analysed several thymic subpopulations for IL-6 production and show that accessory cells, and eventually their precursors, are the major if not exclusive, producers of this cytokine. Mature steroid-resistant thymocytes do not secrete IL-6. Production of IL-6 by total CD4-CD8- thymic cells is largely reduced by the depletion of mature accessory cells which express I-A and Mac-1 antigens. As shown previously, accessory cell precursors within the CD4-CD8- compartment are induced to differentiate into M phi and DC in response to IL-1 and GM-CSF. We provide evidence that this maturation is associated with IL-6 production. Thymic DC and phagocytic cells of the thymic reticulum (P-TR) in vitro produce high levels of IL-6 which are enhanced by GM-CSF or IL-1. These factors have a synergistic effect on IL-6 production by total thymocytes, and on CD4-CD8- cells that are not depleted for mature I-A+ Mac-1+ accessory cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

EMMA-4 analysis of iron in cells of the thymic cortex of a weaver-bird (Quelea quelea).

Combined morphological and analytical studies with the EMMA-4 analytical electron microscope have enabled very early erythroid cells to be identified within the cortex of enlarging thymic lobes of Quelea quelea. These early erythroid cells have pale cytoplasm (sometimes with ferritin-like crystals present), slightly pachychromatic nuclei and have fewer cell organelles (mitochondria) than lymphocytes. Counts for iron were approximately 70% lower than counts from mature erythrocytes found free in the cortex. Iron was also recorded from some epithelial reticular cells and pyknotic nuclei; no iron was recorded from small lymphocytes (thymocytes) in the cortex. The presence of very early erythroid cells is a further indication that erythropoiesis occurs in situ in the avian thymus.     

Macrophages of the rat thymus after cyclosporin treatment

Young adult Wistar rats received 40 mg/kg of cyclosporin perorally for 21 days. Cyclosporin induced almost total disappearance of thymic medulla, whereas the cortex remained preserved. Although the density of cortical macrophages did not change significantly, their characteristics altered markedly and they became enlarged and rounded. In addition to an increase in acid phosphatase and nonspecific esterase activities, cortical macrophages developed very strong succinic dehydrogenase and chloroacetate esterase activities and a fine, granular, aldehyde fuchsinpositive cytoplasmic content. However, these cytoplasmic granules were PAS-negative and were not sudanophilic. Cortical macrophages retained their normal antigenic properties (which were studied by the use of ED1, ED2 and R-MC 41 monoclonal antibodies). Phagocytic cells in the remaining medullary islands retained their usual characteristics. The changes in cortical macrophages after cyclosporin treatment are discussed, especially in relation to the characteristics of macrophages of the cortico-medullary zone in the normal rat thymus.     

Muramyl dipeptide induced augmentation of the proliferative response of thymocytes to phytohemagglutinin

N-Acetylmuramyl-L-alanyl-D-isoglutamine (MDP) augmented the proliferative response of thymocytes to phytohemagglutinin (PHA). The augmenting effect of MDP disappeared by passage of glass-nonadherent thymocytes through Sephadex G-10 (G-10) column or by removal of low density cells by the Ficoll-Conray gradient centrifugation. The diminished augmenting effects of MDP on the proliferative response of glass-nonadherent-G-10 nonadherent thymocytes was restored by the addition of the G-10 adherent cells. When G-10 adherent cell fraction was extensively depleted of macrophages by glass adherence and EA-rosetting, it was found that neither the macrophage-depleted G-10 adherent cell fraction nor the macrophage fraction supported by itself the proliferative response of G-10 nonadherent thymocytes. However, addition of macrophage fraction together with the macrophage-depleted G-10 adherent cells did support the proliferation of G-10 nonadherent thymocytes. It was further shown that peritoneal exudate macrophages could be substituted for thymic macrophage fraction. These results suggested that both the G-10 adherent-glass nonadherent cells and macrophages were essential for the MDP-induced augmentation of the proliferative response of thymocytes to PHA and these cells exerted different accessory roles in this response.     

Naphthol AS D chloroacetate esterase-positive macrophages in the cortico-medullary zone of the normal rat thymus

Naphthol AS D chloroacetate esterase (NASDCE)-positive macrophages are positioned in the cortico-medullary zone (CMZ) of the normal rat thymus. These cells contain the very strongly NASDCE-positive granules of varying size in the cytoplasm. An identical distribution within the thymic parenchyma and an identical morphological appearance is observed in CMZ macrophages after staining with aldehyde fuchsin. The incubation of thymic sections in 10% formalin at pH 7.0 for 48 h does not inhibit the activity of NASDCE in CMZ macrophages. The activity of nonspecific esterase is almost totally abolished by this treatment: only the single, largest globular inclusion within some of the cells remains weakly or moderately positive. The granular content of the CMZ macrophages does not stain metachromatically with toluidine blue and these cells are endogenous peroxidase-negative. NASDCE-positive thymic macrophages are easily distinguished from: a) NASDCE-positive mast-cells, which are confined to the capsular and septal connective tissue and contain densely packed metachromatic granules, and b) NASDCE-positive neutrophilic granulocytes, which have a specific morphological appearance and show very strong endogeneous peroxidatic activity.     

Generation of macrophages from early T progenitors in vitro

Early T progenitors in the thymus have been reported to have the capacity to develop into B cells, thymic dendritic cells, and NK cells. Here we describe conditions that induce early T progenitors to develop into macrophages. Initially, we observed that early T progenitors could be induced to develop into macrophages by cytokines produced from a thymic stromal cell line, TFGD, and later we found that the cytokine mixture of M-CSF plus IL-6 plus IL-7 also induced macrophage differentiation from pro-T cells. M-CSF by itself was unable to induce macrophage differentiation from early T progenitors. To correlate this observation with the developmental potential of early T progenitors, mouse embryonic thymocytes were sorted into four populations, pro-T1 to pro-T4, based on the expression of CD44 and CD25, and then cultured with TFGD culture supernatant. We found that pro-T1 and pro-T2 cells, but not pro-T3 and pro-T4 cells, generate macrophages. Limiting dilution analysis of the differentiation capability of sorted pro-T2 cells also confirmed that pro-T2 cells could generate macrophages. These results suggest that T cells and thymic macrophages could originate from a common intrathymic precursor.     

Stimulator cell requirements for allospecific T cell subsets: specialized accessory cells are required to activate helper but not cytolytic T lymphocyte precursors

Murine cortisone-resistant thymocytes were separated by staining with monoclonal anti-Lyt-2 antibody and FMF into Lyt-2- and Lyt-2+ subsets in order to analyze the nature of stimulator accessory cells required to activate each of these functionally distinct T cell subpopulations. The Lyt-2- fraction was able to proliferate but not to generate cytotoxic cells when stimulated by irradiated allogeneic spleen cells. Fractionation of the stimulator population showed that low numbers of dendritic cells and splenic macrophages, but not equivalent numbers of whole spleen cells or peritoneal macrophages, were able to stimulate the Lyt-2- population. On the other hand, the Lyt-2+ population, which showed little if any proliferation in response to irradiated spleen cells, contained all the precursors of cytolytic T lymphocytes. In contrast to the highly specific stimulator requirement of the Lyt-2- fraction, allospecific cytotoxic cells were generated from Lyt-2+ cells by any alloantigen-bearing stimulator cell provided interleukin 2 was present. This was confirmed by limiting dilution analysis: alloreactive CTL-P frequencies in spleen and thymus were not influenced by the nature of the stimulator cell. These data collectively indicate that heterogeneous Ia+ accessory cells are required to stimulate helper but not cytolytic T cell precursors.     

Effect of capsular polysaccharide of Klebsiella pneumoniae on the differentiation and functional capacity of macrophages cultured in vitro.

The effect of the capsular polysaccharide of Klebsiella pneumoniae (CPS-K) on the differentiation and functional capacity of macrophages cultured in vitro from various lymphoid tissues was investigated. In cultures of peritoneal cells, the number of macrophages did not change throughout incubation periods of from 1 hr to 3 days, and the addition of CPS-K had no affect. It appears therefore that CPS-K does not exhibit cytotoxic effects on macrophages. In cultures of spleen cells, only a small number of macrophages appeared within 1 hr, but the number of macrophages increased during further incubation. The addition of CPS-K to cultures of spleen cells at the start of incubation suppressed markedly the increase in the numbers of macrophage. This finding indicates that CPS-K blocks the process of the generation of macrophages, probably from their precursor cells in cultures of spleen cells. Only a small number of macrophages appeared in cultures of thymocytes or lymph node cells either with or without CPS-K. The phagocytic capacity of either peritoneal macrophages or macrophages generated in cultures of spleen cells was activated during incubation in vitro. Macrophages cultured in the presence of CPS-K for 24 hr or longer appeared to have an enhanced phagocytic activity, although the enhancement of their phagocytic activity by the addition of CPS-K was less marked in cultures of spleen cells than in those of peritoneal macrophages. Morphologically, macrophages in both cultures of peritoneal cells and spleen cells incubated in the presence of CPS-K for 4 days possessed much longer cytoplasmic processes than those incubated in the absence of CPS-K. From the present study, it appears that CPS-K exhibits dual effects on macrophage precursor cells and macrophages, a blocking effect on the differentiation from the former to the latter and an enhancing effect on the functional capacity of the latter.     

Distribution of Lyt antigens on the surface of thymocytes associated with thymic macrophages and dendritic cells

Summary Thymocyte subpopulations that are associated with macrophages and dendritic cells of the thymus in vivo were isolated from the thymuses of C57Bl/6 mice, and their Lyt phenotypes were analyzed. Electron-microscopic examination of immunogold-labeled cells revealed that the thymic complexes formed by macrophages mainly contained Lyt-2-positive thymocytes, while Lyt-1-positive thymocytes were more frequently associated with dendritic cells. The characteristic distributions of Lyt antigens on the surface of thymocytes in regions of reciprocal contact with macrophages (Lyt-2-positive cells) and dendritic cells (Lyt-1-positive cells) suggest that these antigens play a role in specific interactions between thymocytes and stroma cells.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthday